Insulin-like growth factor binding protein, acid labile subunit (IGFALS) compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting the insulin-like growth factor binding protein, acid labile subunit (IGFALS) gene or the insulin-like growth factor 1 (IGF-1) gene, methods of using such double stranded RNAi agents to inhibit expression of an IGFALS gene or an IGF-1 gene, and methods of treating subjects having an IGF system-associated disorder.

RELATED APPLICATION

This application is a 35 U.S.C. § 371 national stage filing ofInternational Application No. PCT/US2016/041440, filed on Jul. 8, 2016,which in turn claims the benefit of priority to U.S. Provisional PatentApplication Nos.: 62/191,008, filed on Jul. 10, 2015; 62/269,401, filedon Dec. 18, 2015; and 62/316,726, filed on Apr. 1, 2016. The entirecontents of each of the foregoing applications are hereby incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Jul. 7, 2016, isnamed 121301-03920_SL.txt and is 1,164,878 bytes in size.

BACKGROUND OF THE INVENTION

Acromegaly is a progressive and life threatening disease resulting fromgrowth hormone hypersecretion from a benign pituitary tumor, leading toapproximately a 10 year reduction in lifespan and a reduced quality oflife. Acromegaly is associated with cardiovascular disease includinghypertension and cardiac hypertrophy, cerebrovascular disease includingstroke, metabolic disease including diabetes, and respiratory diseaseincluding sleep apnea. Mortality rates in acromegaly are correlated withgrowth hormone and IGF-1 levels, with increased growth hormoneconcentrations being associated with shorter life spans (Holdaway etal., JCEM, 2004). The clinical features most commonly associated withacromegaly are acral enlargement, maxofacial changes, excessivesweating, athralgias, headache, hypogonadal symptoms, visual deficit,fatigue, weight gain, and galactorrhea. Such symptoms may be associatedwith any of a number of diseases or conditions and, thus, diagnosis ofacromegaly often does not occur until several years after the initiationof growth hormone hypersecretion. Definitive diagnosis of acromeaglyincludes detection of an increased level of insulin-like growth factor-1(IGF-1) and growth hormone elevation in an oral glucose tolerance test,confirmed by detection of a GH-hypersecreting pituitary tumor, typicallyby MRI. (The diagnostic criteria for acromegaly are provided in theAmerican Association of Clinical Endocrinologists Medical Guidelines forClinical Practice for the Diagnosis and Treatment of Acromegaly-2011Update (Katznelson et al., Endocr. Pract. 17 (Suppl. 4)).

Current treatment options for acromegaly are insufficient for manypatients. Surgical removal of the pituitary adenoma by transsphenoidalsurgery results in a cure for about 50-60% of patients. Subjects forwhom surgical intervention is not possible or does not result in a cureare treated with first-line pharmacological therapy which includesdopamine agonists or sustained-release somatostatin analogs (SSAs). Thistherapy results in good control for the disease for about 70% of thesepatients for whom surgery cannot provide a cure. The use of SSAs,however, is limited to subjects expressing a somatostatin receptor ontheir tumor. Subjects whose disease cannot be controlled by thefirst-line pharmacological therapy are treated with SOMAVERT®(pegvisomant), a growth hormone receptor antagonist, which isadministered by daily subcutaneous injection. Radiotherapy, whichsuffers from low efficacy and high side effects, is used as a lastresort.

The insulin-like growth factor system is also associated with abnormalgrowth in cancer and metastasis (see, e.g., Samani et al., EndocrineRev., 2007). The IGF system has become a target for anticancer agents,both as primary and adjunctive therapy.

Currently, treatments for acromegaly and cancer do not fully meetpatient needs. Therefore, there is a need for therapies for subjectssuffering from acromegaly or cancer.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an insulin-like growth factor binding protein, acidlabile subunit (IGFALS) gene or an insulin-like growth factor-1 (IGF-1)gene. The IGFALS gene or IGF-1 gene may be within a cell, e.g., a cellwithin a subject, such as a human.

In an aspect, the invention provides a double stranded ribonucleic acidinterference (dsRNA) agent for inhibiting expression of insulin-likegrowth factor binding protein, acid labile subunit (IGFALS), wherein thedouble stranded dsRNA agent comprises a sense strand and an antisensestrand, wherein the sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:1 and the antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:2.

In certain embodiments, the sense strands and antisense strands comprisesequences selected from any one of the sequences in any one of Tables 3,5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acidinterference (dsRNAi) agent for inhibiting expression of insulin-likegrowth factor-1 (IGF-1), wherein the double stranded RNAi agentcomprises a sense strand and an antisense strand, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO: 11 or 13and the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO: 12 or 14.

In certain embodiments, the sense strands and antisense strands comprisesequences selected from any one of the sequences in any one of Tables 9,11, 15, 17, 18, or 20.

In an aspect, the invention provides a double stranded ribonucleic acidinterference (dsRNAi) agent for inhibiting expression of insulin-likegrowth factor binding protein, acid labile subunit (IGFALS), wherein thedouble stranded RNAi comprises a sense strand and an antisense strand,the antisense strand comprising a region of complementarity whichcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the antisense sequences listed in any one ofTables 3, 5, 6, 8, 12, or 14.

In an aspect, the invention provides a double stranded ribonucleic acidinterference (dsRNAi) agent for inhibiting expression of insulin-likegrowth factor 1 (IGF-1) wherein the double stranded RNAi comprises asense strand and an antisense strand, the antisense strand comprising aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of theantisense sequences listed in any one of Tables 9, 11, 15, 17, 18, or20.

In certain embodiments, the double stranded RNAi comprises at least onemodified nucleotide. In some embodiments, substantially all of thenucleotides of the sense strand are modified nucleotides. In someembodiments, substantially all of the nucleotides of the antisensestrand are modified nucleotides. In some embodiments, all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand comprise a modification.

In an aspect, the invention provides a double stranded RNAi agent forinhibiting expression of insulin-like growth factor binding protein,acid labile subunit (IGFALS), wherein the double stranded RNAi agentcomprises a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1 and the antisense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:2, wherein substantially allof the nucleotides of the sense strand and substantially all of thenucleotides of the antisense strand are modified nucleotides, andwherein the double stranded RNAi agent comprises a ligand, e.g., thesense strand of the double stranded RNAi agent is conjugated to aligand, e.g., a ligand is attached at the 3′-terminus of the sensestrand.

In an aspect, the invention provides a double stranded ribonucleic acid(RNAi) agent for inhibiting expression of insulin-like growth factor 1(IGF-1), wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 11or 13 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO: 12 or 14, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, and wherein the sensestrand is conjugated to a ligand attached at the 3′-terminus.

Accordingly, in certain embodiments, the present invention providesdouble stranded RNAi agents for inhibiting expression of IGFALS, whichcomprise a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216, 194-229,211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349, 353-371,353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515, 541-580,547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799, 781-799,825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085, 1064-1096,1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163, 1145-1186,1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307, 1321-1339,1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537, 1519-1559,1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635, 1672-1690,1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829, 1871-1889,1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077, 2048-2088, or2052-2084 of SEQ ID NO: 1, and, in certain embodiments, the antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the corresponding position of the nucleotidesequence of SEQ ID NO: 2 such that the antisense strand is complementaryto the at least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

Accordingly, in certain embodiments, the present invention providesdouble stranded RNAi agents for inhibiting expression of insulin-likegrowth factor 1 (IGF-1), which comprise a sense strand and an antisensestrand forming a double stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from nucleotides 330-369, 342-369, 432-490, 432-482,436-462, 534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455,436-456, 438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480,461-481, 462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558,539-559, 542-562, 548-568, 577-597, 582-602, or 640-660 of thenucleotide sequence of SEQ ID NO: 11, and the antisense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the corresponding position of the nucleotide sequenceof SEQ ID NO: 12 such that the antisense strand is complementary to theat least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

In certain embodiments, the present invention provides double strandedRNAi agents for inhibiting expression of insulin-like growth factor 1(IGF-1), which comprise a sense strand and an antisense strand forming adouble stranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480,543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126,1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458,1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825,1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375,2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142,3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704,3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172,4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584,4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902,4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430,5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928,5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612,6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905,6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, and 7252-7270 ofthe nucleotide sequence of SEQ ID NO: 13, and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the corresponding position of the nucleotide sequenceof SEQ ID NO: 14 such that the antisense strand is complementary to theat least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 342-369, 432-462, 330-350, 342-362, 348-368, 349-369,432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490, 481-501,536-556, or 539-559 of the nucleotide sequence of SEQ ID NO: 11 and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the corresponding position of thenucleotide sequence of SEQ ID NO: 12 such that the antisense strand iscomplementary to the at least 15 contiguous nucleotides differing by nomore than 3 nucleotides in the sense strand. In certain embodiments, thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from nucleotides 340-369, 430-490, 430-482,434-460, 532-559, 328-350, 340-362, 346-368, 347-369, 430-452, 433-455,434-456, 436-458, 438-460, 439-461, 440-462, 447-469, 453-475, 458-480,459-481, 460-482, 461-483, 462-484, 468-490, 479-501, 532-554, 534-556,536-558, 537-559, 540-562, 546-568, 575-597, 580-602, or 638-660 of thenucleotide sequence of SEQ ID NO: 11, for example nucleotides 342-369,432-462, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456,438-458, 440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of thenucleotide sequence of SEQ ID NO: 11, and the antisense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the corresponding position of the nucleotide sequenceof SEQ ID NO: 12 such that the antisense strand is complementary to theat least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480,543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126,1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458,1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825,1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375,2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142,3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704,3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172,4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584,4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902,4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430,5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928,5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612,6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905,6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or 7252-7270 ofthe nucleotide sequence of SEQ ID NO: 13, and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the corresponding position of the nucleotide sequenceof SEQ ID NO: 14 such that the antisense strand is complementary to theat least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of thesense strand are modified. In certain embodiments, substantially all ofthe nucleotides of the antisense strand are modified nucleotides. Incertain embodiments, substantially all of the nucleotides of bothstrands are modified. Further, in certain embodiments, the doublestranded RNAi agent comprises a ligand, e.g., the double stranded RNAiagent is conjugated to at least one ligand, wherein the ligand is one ormore GalNAc derivatives attached through a monovalent, a bivalent or atrivalent branched linker.

In certain embodiments, the present invention also provides doublestranded RNAi agents for inhibiting expression of IGFALS, which comprisea sense strand and an antisense strand forming a double stranded region,wherein the sense strand comprises at least 15 contiguous nucleotidesfrom nucleotides 11-62, 24-62, 79-117, 79-130, 155-173, 194-216,194-229, 211-229, 232-293, 254-272, 310-328, 310-349, 324-345, 331-349,353-371, 353-394, 376-394, 407-425, 439-449, 431-470, 484-515, 497-515,541-580, 547-568, 596-647, 616-634, 673-691, 694-712, 694-734, 777-799,781-799, 825-843, 825-855, 869-922, 958-976, 958-988, 1064-1085,1064-1096, 1067-1085, 1067-1096, 1100-1141, 1111-1129, 1145-1163,1145-1186, 1159-1186, 1168-1196, 1168-1214, 1193-1214, 1266-1307,1321-1339, 1342-1373, 1375-1406, 1432-1450, 1454-1472, 1519-1537,1519-1559, 1534-1555, 1541-1559, 1606-1624, 1606-1637, 1613-1635,1672-1690, 1672-1712, 1749-1779, 1783-1801, 1805-1823, 1806-1829,1871-1889, 1871-1919, 1949-1977, 1993-2011, 2013-2042, 2048-2077,2048-2088, or 2052-2084 of the nucleotide sequence of SEQ ID NO:1, andthe antisense strand comprises at least 15 contiguous nucleotides fromthe corresponding position of the nucleotide sequence of SEQ ID NO: 2such that the antisense strand is complementary to the at least 15contiguous nucleotides in the sense strand.

In certain embodiments, the present invention provides double strandedribonucleic acid (RNAi) agent for inhibiting expression of insulin-likegrowth factor 1 (IGF-1), which comprise a sense strand and an antisensestrand forming a double stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides selected from the groupconsisting of nucleotides 330-369, 342-369, 432-490, 432-482, 436-462,534-559, 330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456,438-458, 440-460, 441-461, 442-462, 449-469, 455-475, 460-480, 461-481,462-482, 464-484, 470-490, 484-501, 534-554, 536-556, 538-558, 539-559,542-562, 548-568, 577-597, 582-602, or 640-660 of the nucleotidesequence of SEQ ID NO: 11 and the antisense strand comprises at least 15contiguous nucleotides from the corresponding position of the nucleotidesequence of SEQ ID NO: 12 such that the antisense strand iscomplementary to the at least 15 contiguous nucleotides in the sensestrand.

In certain embodiments, the present invention provides double strandedRNAi agents for inhibiting expression of insulin-like growth factor 1(IGF-1), which comprise a sense strand and an antisense strand forming adouble stranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417, 430-480,543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029, 1075-1126,1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352, 1388-1458,1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727, 1793-1825,1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332, 2357-2375,2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980, 3120-3142,3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603, 3686-3704,3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037, 4154-4172,4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545, 4566-4584,4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843, 4884-4902,4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364, 5409-5430,5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808, 5906-5928,5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535, 6584-6612,6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851, 6858-6905,6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, and 7252-7270 ofthe nucleotide sequence of SEQ ID NO: 13, and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the corresponding position of the nucleotide sequenceof SEQ ID NO: 14 such that the antisense strand is complementary to theat least 15 contiguous nucleotides differing by no more than 3nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides selected from thegroup of nucleotides 342-369, 432-462, 330-350, 342-362, 348-368,349-369, 432-452, 435-455, 436-456, 438-458, 440-460, 442-462, 470-490,481-501, 536-556, or 539-559 of the nucleotide sequence of SEQ ID NO:11and the antisense strand comprises at least 15 contiguous nucleotidesfrom the corresponding position of the nucleotide sequence of SEQ ID NO:12 such that the antisense strand is complementary to the at least 15contiguous nucleotides in the sense strand.

In certain embodiments, the agents comprise a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides selected from thegroup of nucleotides 340-369, 430-490, 430-482, 434-460, 532-559,328-350, 340-362, 346-368, 347-369, 430-452, 433-455, 434-456, 436-458,438-460, 439-461, 440-462, 447-469, 453-475, 458-480, 459-481, 460-482,461-483, 462-484, 468-490, 479-501, 532-554, 534-556, 536-558, 537-559,540-562, 546-568, 575-597, 580-602, or 638-660 of the nucleotidesequence of SEQ ID NO: 11, for example nucleotides 342-369, 432-462,330-350, 342-362, 348-368, 349-369, 432-452, 435-455, 436-456, 438-458,440-460, 442-462, 470-490, 481-501, 536-556, or 539-559 of thenucleotide sequence of SEQ ID NO: 11, and the antisense strand comprisesat least 15 contiguous nucleotides from the corresponding position ofthe nucleotide sequence of SEQ ID NO: 12 such that the antisense strandis complementary to the at least 15 contiguous nucleotides in the sensestrand.

In certain embodiments, the agents comprise a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides selected from thegroup of nucleotides 6-90, 127-145, 185-238, 247-265, 277-295, 389-417,430-480, 543-561, 654-690, 750-768, 774-870, 894-930, 1007-1029,1075-1126, 1144-1162, 1197-1215, 1232-1250, 1293-1311, 1334-1352,1388-1458, 1463-1490, 1511-1529, 1599-1617, 1643-1661, 1690-1727,1793-1825, 1843-1861, 2057-2075, 2090-2130, 2192-2228, 2310-2332,2357-2375, 2521-2539, 2566-2588, 2648-2684, 2793-2811, 2962-2980,3120-3142, 3208-3233, 3269-3287, 3417-3435, 3449-3467, 3575-3603,3686-3704, 3721-3739, 3806-3824, 3939-3957, 3982-4018, 4081-4037,4154-4172, 4271-4289, 4319-4377, 4436-4478, 4484-4502, 4523-4545,4566-4584, 4610-4660, 4686-4717, 4734-4769, 4780-4798, 4815-4843,4884-4902, 4911-4929, 5004-5034, 5050-5068, 5171-5256, 5311-5364,5409-5430, 5551-5588, 5609-5638, 5694-5712, 5715-5758, 5790-5808,5906-5928, 5934-5952, 6323-6345, 6399-6417, 6461-6497, 6510-6535,6584-6612, 6629-6647, 6661-6683, 6726-6789, 6796-6824, 6826-6851,6858-6905, 6910-6927, 7004-7022, 7035-7130, 7144-7162, 7175-7241, or7252-7270 of the nucleotide sequence of SEQ ID NO: 13, and the antisensestrand comprises at least 15 contiguous nucleotides from thecorresponding position of the nucleotide sequence of SEQ ID NO: 14 suchthat the antisense strand is complementary to the at least 15 contiguousnucleotides in the sense strand.

In certain embodiments, substantially all of the nucleotides of thesense strand are modified nucleotides. In certain embodiments,substantially all of the nucleotides of the antisense strand aremodified nucleotides. In certain embodiments, substantially all of thenucleotides of both strands are modified. In preferred embodiments, thedouble stranded RNAi agent comprises a ligand, e.g., the double strandedRNAi agent is conjugated to at least one ligand, wherein the ligand isone or more GalNAc derivatives attached through a monovalent, a bivalentor a trivalent branched linker.

In certain embodiments, the sense strand and the antisense strandcomprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense sequences listed in any one of Tables 3, 5, 6, 8,12, or 14 for IGFALS or any one of Tables 9, 11, 15, 17, 18, or 20 forIGF-1.

For example, in certain embodiments, the sense strand and the antisensestrand comprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense nucleotide sequences selected from the group of theantisense nucleotide sequence of duplexes targeted to IGF selected fromthe group AD-66722, AD-66748, AD-66746, AD-66747, AD-66733, AD-66752,AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, AD-66745,AD-66749, AD-66720, AD-66724, AD-66726, AD-66766, AD-66761, AD-66755,AD-66751, AD-66719, AD-66727, AD-66744, AD-66760, AD-66753, AD-66721,AD-66716, AD-66743, or AD-66728, AD-77150, AD-77158, AD-74963, AD-77138,AD-75740, AD-74968, AD-74965, AD-75766, AD-75761, AD-75137, AD-74979,AD-74966, AD-75750, AD-77126, AD-74971, AD-74982, AD-77144, AD-77149,AD-75751, AD-75111, AD-77147, AD-74964, AD-74983, AD-75765, AD-74970,AD-75749, AD-77168, AD-77127, AD-75748, AD-75779, AD-75145, AD-74975,AD-77151, AD-75170, AD-75741, AD-75162, AD-74985, AD-75759, AD-75218,AD-74981, AD-75155, AD-74978, AD-77153, AD-75157, AD-75123, AD-75184,AD-77160, AD-75125, AD-75229, AD-77165, AD-75112, AD-75206, AD-75769,AD-75174, AD-75225, AD-75792, AD-75115, AD-74986, AD-77171, AD-75131,AD-77128, AD-75179, AD-75792, AD-77124, AD-75191, AD-75774, AD-75114,AD-74973, AD-77156, AD-75120, AD-75130, AD-74967, AD-75231, AD-74987,AD-77140, AD-74969, AD-75000, AD-75791, AD-75143, AD-77120, AD-77142,AD-75217, AD-75234, AD-75173, AD-75232, AD-75188, AD-75135, AD-75018,AD-77122, AD-75009, AD-75121, AD-75791, AD-77135, AD-75214, AD-74994,AD-75139, AD-75166, AD-75020, AD-77159, AD-75236, AD-77123, AD-77133,AD-74972, AD-75223, AD-75148, AD-75124, AD-75185, AD-75150, AD-74976,AD-74980, AD-75212, AD-75239, AD-75221, AD-75118, AD-75793, AD-75023,AD-75164, AD-74997, AD-74984, AD-75011, AD-75203, AD-77161, AD-75033,AD-75177, AD-75795, AD-77146, AD-75793, AD-75788, AD-75079, AD-75152,AD-77121, AD-75237, AD-75014, AD-75755, AD-75028, AD-75091, AD-75110,AD-75230, AD-75029, AD-75099, AD-77130, AD-75224, AD-75142, AD-75760,AD-75795, AD-77136, AD-75032, AD-75757, AD-75017, AD-75151, AD-75122,AD-75002, AD-75021, AD-75005, AD-75088, AD-75153, AD-75208, AD-74977,AD-75069, AD-75107, AD-74990, AD-75061, AD-75083, AD-75116, AD-75169,AD-75058, AD-74991, AD-75041, AD-77131, AD-75772, AD-77169, AD-75133,AD-75222, AD-75007, AD-75101, AD-77137, AD-75090, AD-77148, AD-75008,AD-77134, AD-74999, AD-75048, AD-75095, AD-74974, AD-75788, AD-75057,AD-75113, AD-77172, AD-75016, AD-75186, AD-75205, AD-75238, or AD-75146;for example duplexes AD-66722, AD-66748, AD-66746, AD-66747, AD-66733,AD-66752, AD-66739, AD-66738, AD-66725, AD-66740, AD-66750, AD-66729, orAD-66745. In certain embodiments, nucleotide sequences selected from thegroup duplexes targeted to IGF selected from the group AD-66722,AD-66748, AD-66746, AD-66747, AD-66733, AD-66752, AD-66739, AD-66738,AD-66725, AD-66740, AD-66750, AD-66729, and AD-66745. In certainembodiments, the sense strand and the antisense strand comprise a regionof complementarity which comprises at least 15 contiguous nucleotides ofany one of the sense and antisense nucleotide sequences of the foregoingduplexes.

In certain embodiments, the sense strand and the antisense strandcomprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense sequences of the duplexes targeted to IGFALSselected from the group AD-62728, AD-62734, AD-68111, AD-68709,AD-68712, AD-68715, AD-68716, AD-68717, AD-68719, AD-68720, AD-68722,AD-68725, AD-68726, AD-68730, AD-68731, AD-73782, AD-73773, AD-73765,AD-73946, AD-73947, AD-73858, AD-73797, AD-73808, AD-73906, AD-73912,AD-73848, AD-73836, AD-73818, AD-73786, AD-73862, AD-73795, AD-73766,AD-73930, AD-73825, AD-73924, AD-73802, AD-73767, AD-73771, AD-73777,AD-73793, AD-73898, AD-73784, AD-73882, AD-73803, AD-73772, AD-73907,AD-73948, AD-73890, AD-73883, AD-73770, AD-73867, AD-73931, AD-73932,AD-73787, AD-73791, AD-73880, AD-73914, AD-73849, AD-73863, AD-73920,AD-73944, AD-73841, AD-73785, AD-73804, AD-73823, AD-73885, AD-73788,AD-73865, AD-73941, AD-73859, AD-73913, AD-73892, AD-73837, AD-73842,AD-73840, AD-73813, AD-73796, AD-73875, AD-73900, AD-73922, AD-73861,AD-73816, AD-73764, AD-73868, AD-73812, AD-73826, AD-73938, AD-73843,AD-73817, AD-73943, AD-73827, AD-73937, AD-73877, AD-73833, AD-73807,AD-73819, AD-73886, AD-73919, AD-73800, AD-76171, AD-76173, AD-76203,AD-76210, AD-76172, AD-76175, AD-76209, AD-76174, AD-76208, AD-76186,AD-76177, AD-76199, AD-76197, or AD-76212.

In certain embodiments, substantially all of the nucleotides of thesense strand are modified nucleotides. In certain embodiments,substantially all of the nucleotides of the antisense strand aremodified nucleotides. In certain embodiments, substantially all of thenucleotides of both strands are modified.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a deoxy-nucleotide, a 3′-terminaldeoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitolmodified nucleotide, a cyclohexenyl modified nucleotide, a nucleotidecomprising a phosphorothioate group, a nucleotide comprising amethylphosphonate group, a nucleotide comprising a 5′-phosphate, and anucleotide comprising a 5′-phosphate mimic. In another embodiment, themodified nucleotides comprise a short sequence of 3′-terminaldeoxy-thymine nucleotides (dT).

In certain embodiments, substantially all of the nucleotides of thesense strand are modified. In certain embodiments, substantially all ofthe nucleotides of the antisense strand are modified. In certainembodiments, substantially all of the nucleotides of both the sensestrand and the antisense strand are modified.

In certain embodiments, the duplex comprises a modified antisensenucleotide sequence targeted to IGFALS provided in Table 5, 8, or 14, ortargeted to IGF-1 in Table 11, 17, or 20. In certain embodiments, theduplex comprises a modified sense strand nucleotide sequence targeted toIGFALS provided in Table 5, 8, or 14, or targeted to IGF-1 in Table 11,17, or 20. In certain embodiments, the duplex comprises the modifiedsense strand nucleotide sequence and the modified antisense strandnucleotide of any one of the duplexes targeted to IGFALS provided inTable 5, 8, or 14, or targeted to IGF-1 in Table 11, 17, or 20.

In certain embodiments, the region of complementarity between theantisense strand and the target is at least 17 nucleotides in length.For example, the region of complementarity between the antisense strandand the target is 19 to 21 nucleotides in length, for example, theregion of complementarity is 21 nucleotides in length. In preferredembodiments, each strand is no more than 30 nucleotides in length.

In some embodiments, at least one strand comprises a 3′ overhang of atleast 1 nucleotide, e.g., at least one strand comprises a 3′ overhang ofat least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14,or 15 nucleotides. In other embodiments, at least one strand of the RNAiagent comprises a 5′ overhang of at least 1 nucleotide. In certainembodiments, at least one strand comprises a 5′ overhang of at least 2nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15nucleotides. In still other embodiments, both the 3′ and the 5′ end ofone strand of the RNAi agent comprise an overhang of at least 1nucleotide. In other embodiment, at least one strand comprises a 3′overhang of at least 2 nucleotides.

In many embodiments, the double stranded RNAi agent further comprises aligand. The ligand may be one or more GalNAc attached to the RNAi agentthrough a monovalent, a bivalent, or a trivalent branched linker. Theligand may be conjugated to the 3′ end of the sense strand of the doublestranded RNAi agent. The ligand can be an N-acetylgalactosamine (GalNAc)derivative including, but not limited to

In various embodiments, the ligand is attached to the 5′ end of thesense strand of the double stranded RNAi agent, the 3′ end of theantisense strand of the double stranded RNAi agent, or the 5′ end of theantisense strand of the double stranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the inventioncomprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, eachindependently attached to a plurality of nucleotides of the doublestranded RNAi agent through a plurality of monovalent linkers.

In certain embodiments, the dsRNAi is agent conjugated to the ligand asshown in the following schematic:

and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the ligand is a cholesterol.

In certain embodiments, the region of complementarity comprises any oneof the antisense sequences targeted to IGFALS provided in Table 3, 5, 6,8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20. Inanother embodiment, the region of complementarity consists of any one ofthe antisense sequences of targeted to IGFALS provided in Table 3, 5, 6,8, 12, or 14 or targeted to IGF-1 in Table 9, 11, 15, 17, 18, or 20.

In another aspect, the invention provides a double stranded RNAi agentfor inhibiting expression of IGFALS or IGF-1, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding IGFALS or IGF-1, wherein eachstrand is about 14 to about 30 nucleotides in length, wherein the doublestranded RNAi agent is represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present, independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe double stranded RNAi agent comprises a ligand, e.g., the sensestrand is conjugated to at least one ligand.

In certain embodiments, i is 0; j is 0; i is 1; j is 1; both i and j are0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1;l is 1; both k and l are 0; or both k and l are 1. In anotherembodiment, XXX is complementary to X′X′X′, YYY is complementary toY′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In another embodiment, theYYY motif occurs at or near the cleavage site of the sense strand. Inanother embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13positions of the antisense strand from the 5′-end. In one embodiment,the Y′ is 2′-O-methyl.

For example, formula (III) can be represented by formula (IIIa):sense: 5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′antisense: 3′n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′)5′  (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):sense: 5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′antisense: 3′n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′)5′  (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

Alternatively, formula (III) can be represented by formula (IIIc):sense: 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′antisense: 3′n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(a′)-n_(q′)5′  (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

Further, formula (III) can be represented by formula (IIId):sense: 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′antisense:3′n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′)5′  (IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

In certain embodiment, the double stranded region is 15-30 nucleotidepairs in length. For example, the double stranded region can be 17-23nucleotide pairs in length. The double stranded region can be 17-25nucleotide pairs in length. The double stranded region can be 23-27nucleotide pairs in length. The double stranded region can be 19-21nucleotide pairs in length. The double stranded region can be 21-23nucleotide pairs in length.

In certain embodiments, each strand has 15-30 nucleotides. In otherembodiments, each strand has 19-30 nucleotides.

Modifications on the nucleotides are selected from the group including,but not limited to, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl,2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, andcombinations thereof. In another embodiment, the modifications on thenucleotides are 2′-O-methyl or 2′-fluoro modifications.

In many embodiments, the double stranded RNAi agent further comprises aligand. The ligand may be one or more GalNAc attached to the RNAi agentthrough a monovalent, a bivalent, or a trivalent branched linker. Theligand may be conjugated to the 3′ end of the sense strand of the doublestranded RNAi agent. The ligand can be an N-acetylgalactosamine (GalNAc)derivative including, but not limited to

In various embodiments, the ligand is attached to the 5′ end of thesense strand of the double stranded RNAi agent, the 3′ end of theantisense strand of the double stranded RNAi agent, or the 5′ end of theantisense strand of the double stranded RNAi agent.

In some embodiments, the double stranded RNAi agents of the inventioncomprise a plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, eachindependently attached to a plurality of nucleotides of the doublestranded RNAi agent through a plurality of monovalent linkers.

An exemplary structure of a dsRNAi agent conjugated to the ligand isshown in the following schematic

In certain embodiments, the ligand can be a cholesterol.

In certain embodiments, the double stranded RNAi agent further comprisesat least one phosphorothioate or methylphosphonate internucleotidelinkage. For example the phosphorothioate or methylphosphonateinternucleotide linkage can be at the 3′-terminus of one strand, i.e.,the sense strand or the antisense strand; or at the ends of bothstrands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand, i.e., thesense strand or the antisense strand; or at the ends of both strands,the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand, i.e., the sense strand or the antisense strand; or at the endsof both strands, the sense strand and the antisense strand.

In certain embodiments, the base pair at the 1 position of the 5′-end ofthe antisense strand of the duplex is an AU base pair.

In certain embodiments, the Y nucleotides contain a 2′-fluoromodification. In another embodiment, the Y′ nucleotides contain a2′-O-methyl modification. In another embodiment, p′>0. In someembodiments, p′=2. In some embodiments, q′=0, p=0, q=0, and p′ overhangnucleotides are complementary to the target mRNA. In some embodiments,q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to thetarget mRNA.

In certain embodiments, the sense strand has a total of 21 nucleotidesand the antisense strand has a total of 23 nucleotides.

In certain embodiments, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage. In other embodiments, alln_(p)′ are linked to neighboring nucleotides via phosphorothioatelinkages.

In certain embodiments, the dsRNAi agent is selected from the group ofany one of the double stranded RNAi agents targeted to IGFALS providedin Table 3, 5, 6, 8, 12, or 14, or targeted to IGF-1 in Table 9, 11, 15,17, 18, or 20. In certain embodiments, all of the nucleotides of thesense strand and all of the nucleotides of the antisense strand comprisea modification.

In an aspect, the invention provides a double stranded RNAi agent forinhibiting expression of IGFALS or IGF-1 in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding IGFALS or IGF-1, wherein eachstrand is about 14 to about 30 nucleotides in length, wherein the doublestranded RNAi agent is represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; modifications on N_(b) differ from the modification on Yand modifications on N_(b)′ differ from the modification on Y′; andwherein the double stranded RNAi agent comprises a ligand, e.g., thedouble stranded RNAi agent is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through amonovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent forinhibiting expression of IGFALS or IGF-1 in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding IGFALS or IGF-1, wherein eachstrand is about 14 to about 30 nucleotides in length, wherein the doublestranded RNAi agent is represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6; n_(p)′>0 and at least onen_(p)′ is linked to a neighboring nucleotide via a phosphorothioatelinkage; each N_(a) and N_(a)′ independently represents anoligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides; each N_(b) and N_(b)′independently represents an oligonucleotide sequence comprising 0-10nucleotides which are either modified or unmodified or combinationsthereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independentlyrepresent one motif of three identical modifications on threeconsecutive nucleotides, and wherein the modifications are 2′-O-methylor 2′-fluoro modifications; modifications on N_(b) differ from themodification on Y and modifications on N_(b)′ differ from themodification on Y′; and wherein the double stranded RNAi agent comprisesa ligand, e.g., the double stranded RNAi agent is conjugated to at leastone ligand, wherein the ligand is one or more GalNAc derivativesattached through a monovalent, a bivalent or a trivalent branchedlinker.

In certain embodiments, the invention provides a d double strandedribonucleic acid (RNAi) agent for inhibiting expression of IGFALS orIGF-1, wherein the double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein the antisense strandcomprises a region complementary to part of an mRNA encoding IGFALS orIGF-1, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein i, j, k, and l are each independently 0 or 1; each n_(p), n_(q),and n_(q)′, each of which may or may not be present, independentlyrepresents an overhang nucleotide; p, q, and q′ are each independently0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe double stranded RNAi agent is conjugated to at least one ligand,wherein the ligand is one or more GalNAc derivatives attached through amonovalent, a bivalent or a trivalent linker.

In an aspect, the invention provides a double stranded RNAi agent forinhibiting expression of IGFALS or IGF-1, wherein the double strandedRNAi agent comprises a sense strand complementary to an antisensestrand, wherein the antisense strand comprises a region complementary topart of an mRNA encoding IGFALS or IGF-1, wherein each strand is about14 to about 30 nucleotides in length, wherein the double stranded RNAiagent is represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein i, j, k, and l are each independently 0 or 1; each n_(p), n_(q),and n_(q)′, each of which may or may not be present, independentlyrepresents an overhang nucleotide; p, q, and q′ are each independently0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; wherein thedouble stranded RNAi agent comprises a ligand, e.g., the double strandedRNAi agent is conjugated to at least one ligand, wherein the ligand isone or more GalNAc derivatives attached through a monovalent, a bivalentor a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agentcapable of inhibiting the expression of IGFALS or IGF-1 in a cell,wherein the double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein the antisense strandcomprises a region complementary to part of an mRNA encoding IGFALS ofIGF-1, wherein each strand is about 14 to about 30 nucleotides inlength, wherein the double stranded RNAi agent is represented by formula(III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein i, j, k, and l are each independently 0 or 1; each n_(p), n_(q),and n_(q)′, each of which may or may not be present, independentlyrepresents an overhang nucleotide; p, q, and q′ are each independently0-6; n_(p)′>0 and at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; wherein thesense strand comprises at least one phosphorothioate linkage; andwherein the double stranded RNAi agent comprises a ligand, e.g., thedouble stranded RNAi agent is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through amonovalent, a bivalent or a trivalent branched linker.

In an aspect, the invention provides a double stranded RNAi agent forinhibiting expression of IGFALS or IGF-1 in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding IGFALS or IGF-1, wherein eachstrand is about 14 to about 30 nucleotides in length, wherein the doublestranded RNAi agent is represented by formula (III):sense: 5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′antisense: 3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a′)-n_(q′)5′  (IIIa).

wherein each n_(p), n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide; p, q, and q′are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via a phosphorothioate linkage; each N_(a)and N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 nucleotides which are either modified or unmodified orcombinations thereof, each sequence comprising at least two differentlymodified nucleotides; YYY and Y′Y′Y′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; wherein the sense strand comprises at least onephosphorothioate linkage; wherein the double stranded RNAi agentcomprises a ligand, e.g., the double stranded RNAi agent is conjugatedto at least one ligand, wherein the ligand is one or more GalNAcderivatives attached through a monovalent, a bivalent or a trivalentbranched linker.

In an aspect, the invention provides a double stranded ribonucleic acid(RNAi) agent for inhibiting expression of IGFALS, wherein the doublestranded RNAi agent comprises a sense strand and an antisense strandforming a double stranded region, wherein the sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of SEQ ID NO:1 and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2, whereinsubstantially all of the nucleotides of the sense strand comprise amodification selected from a 2′-O-methyl modification and a 2′-fluoromodification, wherein the sense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus, wherein substantially allof the nucleotides of the antisense strand comprise a modificationselected from a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha monovalent, a bivalent or a trivalent linker at the 3′-terminus.

In an aspect, the invention provides a double stranded ribonucleic acid(RNAi) agent for inhibiting expression of insulin-like growth factor 1(IGF-1), wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:11or 13 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:12 or 14, wherein substantially all of thenucleotides of the sense strand comprise a modification selected from a2′-O-methyl modification and a 2′-fluoro modification, wherein the sensestrand comprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from a 2′-O-methylmodification and a 2′-fluoro modification, wherein the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and wherein the sense strand is conjugated to one or moreGalNAc derivatives attached through a monovalent, a bivalent or atrivalent linker at the 3′-terminus.

In certain embodiments, all of the nucleotides of the sense strand andall of the nucleotides of the antisense strand are modified nucleotides.In certain embodiments, each strand has 19-30 nucleotides.

In certain embodiments, substantially all of the nucleotides of thesense strand are modified. In certain embodiments, substantially all ofthe nucleotides of the antisense strand are modified. In certainembodiments, substantially all of the nucleotides of both the sensestrand and the antisense strand are modified.

In an aspect, the invention provides a cell containing the dsRNAi agentas described herein.

In an aspect, the invention provides a vector encoding at least onestrand of a dsRNAi agent, wherein the RNAi agent comprises a region ofcomplementarity to at least a part of an mRNA encoding IGFALS or IGF-1,wherein the RNAi is 30 base pairs or less in length, and wherein theRNAi agent targets the mRNA for cleavage. In certain embodiments, theregion of complementarity is at least 15 nucleotides in length. Incertain embodiments, the region of complementarity is 19 to 23nucleotides in length.

In an aspect, the invention provides a cell comprising a vector asdescribed herein.

In an aspect, the invention provides a pharmaceutical composition forinhibiting expression of an IGFALS or IGF-1 gene, comprising a doublestranded RNAi agent of the invention. In one embodiment, the RNAi agentis administered in an unbuffered solution. In certain embodiments, theunbuffered solution is saline or water. In other embodiments, the RNAiagent is administered with a buffer solution. In such embodiments, thebuffer solution can comprise acetate, citrate, prolamine, carbonate, orphosphate, or any combination thereof. For example, the buffer solutioncan be phosphate buffered saline (PBS).

In an aspect, the invention provides a pharmaceutical compositioncomprising the double stranded RNAi agent of the invention and a lipidformulation. In certain embodiments, the lipid formulation comprises aLNP. In certain embodiments, the lipid formulation comprises MC3.

In an aspect, the invention provides a method of inhibiting IGFALS orIGF-1 expression in a cell, the method comprising (a) contacting thecell with the double stranded RNAi agent of the invention or apharmaceutical composition of the invention; and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of an IGFALS or IGF-1 gene, thereby inhibitingexpression of the IGFALS or IGF-1 gene in the cell. In certainembodiments, the cell is within a subject, for example, a human subject,for example a female human or a male human. In preferred embodiments,IGFALS or IGF-1 expression is inhibited by at least 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%, or to below the threshold of detection of theassay method used. Preferably the expression is inhibited by at least50%. In some embodiments of the methods of the invention, expression ofan IGF-1 gene is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the difference between the elevated level associated withthe disease and a normal level in an appropriate control subject.Preferably the elevated level is inhibited by at least 50%.

In an aspect, the invention provides a method of treating a subjecthaving a disease or disorder that would benefit from reduction in IGFALSor IGF-1 expression, such as an IGF system-associated disease ordisorder, the method comprising administering to the subject atherapeutically effective amount of a double stranded RNAi agent of theinvention or a pharmaceutical composition of the invention, therebytreating the subject.

In an aspect, the invention provides a method of preventing at least onesymptom in a subject having a disease or disorder that would benefitfrom reduction in IGFALS or IGF-1 expression, such as an IGFsystem-associated disease or disorder, the method comprisingadministering to the subject a prophylactically effective amount of adouble stranded RNAi agent of the invention or a pharmaceuticalcomposition of the invention, thereby preventing at least one symptom inthe subject having a disorder that would benefit from reduction inIGFALS or IGF-1 expression.

In certain embodiments, the administration of the double stranded RNAito the subject causes a decrease in the IGF-1 signaling pathway. Incertain embodiments, the administration of the double stranded RNAicauses a decrease in the level of IGF-1 or IGFALS in the subject, e.g.,serum levels of IGF-1 or IGFALS in the subject.

In certain embodiments, the IGF system-associated disease is acromegaly.In certain embodiments, the IGF system-associated disease is gigantism.In another embodiment, the IGF system-associated disease is cancer. Incertain embodiments, the cancer is metastatic cancer.

In certain embodiments, the invention further comprises administering aninhibitor of growth hormone to a subject with an IGF system-associateddisease.

In certain embodiments, the invention further comprises administering aninhibitor of the IGF pathway signaling to a subject with an IGFsystem-associated disease.

In certain embodiments, wherein the IGF system-associated disease isacromegaly or gigantism, the subject is further treated for acromegalyor gigantism. In certain embodiments, the treatment for acromegaly orgigantism includes surgery. In certain embodiments, the treatment foracromegaly or gigantism includes radiation. In certain embodiments, thetreatment for acromegaly or gigantism includes administration of atherapeutic agent.

In certain embodiments, wherein the IGF system-associated disease iscancer, the subject is further treated for cancer. In certainembodiments, the treatment for cancer includes surgery. In certainembodiments, the treatment for cancer includes radiation. In certainembodiments, the treatment for cancer includes administration of achemotherapeutic agent.

In various embodiments, the dsRNAi agent is administered at a dose ofabout 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.In some embodiments, the dsRNAi agent is administered at a dose of about10 mg/kg to about 30 mg/kg. In certain embodiments, the dsRNAi agent isadministered at a dose selected from 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, thedsRNAi agent is administered about once per week, once per month, onceevery other two months, or once a quarter (i.e., once every threemonths) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.

In certain embodiments, the double stranded RNAi agent is administeredto the subject once a week. In certain embodiments, the dsRNAi agent isadministered to the subject once a month. In certain embodiments, thedsRNAi agent is administered once per quarter (i.e., every threemonths).

In some embodiment, the dsRNAi agent is administered to the subjectsubcutaneously.

In various embodiments, the methods of the invention further comprisedetermining the level of IGF-1 in the subject. In certain embodiments, adecrease in the level of expression or activity of the IGF-1 signalingpathway indicates that the IGF system-associated disease is beingtreated.

In various embodiments, a surrogate marker of IGF-1 expression ismeasured. In certain embodiments, a change, preferably a clinicallyrelevant change in the surrogate marker indicating effective treatmentof diseases associated with an elevated IGF-level are detected, e.g.,decreased serum IGF. In the treatment of acromegaly, a clinicallyrelevant change in one or more signs or symptoms associated withacromegaly as provided below can be used as a surrogate marker for areduction in IGF-1 expression. In the treatment of cancer, ademonstration of stabilization or reduction of tumor burden using RECISTcriteria can be used as a surrogate marker for a reduction of IGF-1expression or activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing various aspects of the IGF-1 signalingpathways.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an Insulin-like Growth Factor Binding Protein, AcidLabile Subunit (IGFALS) or Insulin-like Growth Factor 1 (IGF-1) gene.The gene may be within a cell, e.g., a cell within a subject, such as ahuman. The use of these iRNAs enables the targeted degradation of mRNAsof the corresponding gene (IGFALS or IGF-1 gene) in mammals.

The iRNAs of the invention have been designed to target a human IGFALSor a human IGF-1 gene, including portions of the gene that are conservedin the IGFALS or IGF-1 othologs of other mammalian species. Withoutintending to be limited by theory, it is believed that a combination orsub-combination of the foregoing properties and the specific targetsites or the specific modifications in these iRNAs confer to the iRNAsof the invention improved efficacy, stability, potency, durability, andsafety.

Accordingly, the present invention also provides methods for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an IGFALS or IGF-1 gene, e.g., an IGFsystem-associated disease, such as acromegaly or cancer, such as acancer in which the tumor expresses IGF-1, using iRNA compositions whicheffect the RNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an IGFALS or an IGF-1 gene.

Very low dosages of the iRNAs of the invention, in particular, canspecifically and efficiently mediate RNA interference (RNAi), resultingin significant inhibition of expression of the corresponding target gene(IGFALS or IGF-1gene).

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan IGFALS or IGF-1 gene.

In certain embodiments, the iRNAs of the invention include an RNA strand(the antisense strand) which can include longer lengths, for example upto 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53nucleotides in length with a region of at least 19 contiguousnucleotides that is substantially complementary to at least a part of anmRNA transcript of an IGFALS or an IGF-1 gene.

In some embodiments, the iRNA agents for use in the methods of theinvention include an RNA strand (the antisense strand) which can be upto 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43,27-53 nucleotides in length, with a region of at least 19 contiguousnucleotides that is substantially complementary to at least a part of anmRNA transcript of an IGFALS or an IGF-1 gene. In some embodiments, suchiRNA agents having longer length antisense strands preferably include asecond RNA strand (the sense strand) of 20-60 nucleotides in lengthwherein the sense and antisense strands form a duplex of 18-30contiguous nucleotides.

Using in vitro and in vivo assays, the present inventors havedemonstrated that iRNAs targeting an IGFALS gene or an or IGF-1 gene canmediate RNAi, resulting in significant inhibition of expression ofIGFALS or IGF-1, as well as reducing signaling through the IGF-1 pathwaywhich will decrease one or more of the symptoms associated with an IGFsystem-associated disease, such as acromegaly or cancer. Thus, methodsand compositions including these iRNAs are useful for treating a subjecthaving an IGF system-associated disease, such as acromegaly or cancer.The methods and compositions herein are useful for reducing the level ofIGFALS or IGF-1 in a subject, e.g., serum or liver IGF-1 in a subject,especially in a subject with acromegaly or a tumor, such as an IGF-1expressing tumor.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of an IGFALSgene or an IGF gene as well as compositions, uses, and methods fortreating subjects having diseases and disorders that would benefit fromreduction of the expression of an IGFALS gene or an IGF gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise. Forexample, “sense strand or antisense strand” is understood as “sensestrand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, aboutmeans±10%. In certain embodiments, about means±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understoodto include the number adjacent to the term “at least”, and allsubsequent numbers or integers that could logically be included, asclear from context. For example, the number of nucleotides in a nucleicacid molecule must be an integer. For example, “at least 18 nucleotidesof a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21nucleotides have the indicated property. When at least is present beforea series of numbers or a range, it is understood that “at least” canmodify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the valueadjacent to the phrase and logical lower values or intergers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range.

As used herein, ranges include both the upper and lower limit.

In the event of a conflict between a sequence and its indicated site ona transcript or other sequence, the nucleotide sequence recited in thespecification takes precedence.

Various embodiments of the invention can be combined as determinedappropriate by one of skill in the art.

As used herein, “insulin-like growth factor binding protein, acid labilesubunit” or “IGFALS” is a serum protein that binds insulin-like growthfactors, increasing their half-life and their vascular localization.Production of the encoded protein, predominantly in the liver, whichcontains twenty leucine-rich repeats, is stimulated by growth hormone.Defects in this gene are a cause of acid-labile subunit deficiency,which manifests itself in delayed and slow puberty. Three transcriptvariants encoding two different isoforms have been found for this gene.The gene can also be known as ALS or ACLSD. Further information onIGFALS is provided, for example in the NCBI Gene database atwww.ncbi.nlm.nih.gov/gene/3483 (which is incorporated herein byreference as of the date of filing this application).

As used herein, “insulin-like growth factor binding protein, acid labilesubunit,” used interchangeably with the term “IGFALS,” refers to thenaturally occurring gene that encodes an IGF-1 binding protein. Theamino acid and complete coding sequences of the reference sequence ofthe human IGFALS gene may be found in, for example, GenBank AccessionNo. GI: 225579150 (RefSeq Accession No. NM_004970.2; SEQ ID NO:1; SEQ IDNO:2), GenBank Accession No. GI:225579151 (RefSeq Accession No.NM_001146006.1; SEQ ID NO: 9 and 10). Mammalian orthologs of the humanIGFALS gene may be found in, for example, GI:142388344 (RefSeq AccessionNo. NM_008340.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:71896591(RefSeq Accession No. NM_053329.2, rat; SEQ ID NO:5 and SEQ ID NO:6);GenBank Accession Nos. GI:544514850 (RefSeq Accession No.XM_005590898.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8).

A number of naturally occurring SNPs are known and can be found, forexample, in the SNP database at the NCBI atwww.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=3483 (which is incorporatedherein by reference as of the date of filing this application) whichlists SNPs in human IGFALS. In preferred embodiments, such naturallyoccurring variants are included within the scope of the IGFALS genesequence.

Additional examples of IGFALS mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, and OMIM.

“Insulin-like growth factor 1” or “IGF-1”, also known as MGF, encodes aprotein similar to insulin in function and structure and is a member ofa family of proteins involved in mediating growth and development. Theencoded protein is processed from a precursor, bound by a specificreceptor, and secreted. Defects in this gene are a cause of insulin-likegrowth factor I deficiency. Alternative splicing results in multipletranscript variants encoding different isoforms that may undergo similarprocessing to generate mature protein. Further information on IGF-1 isprovided, for example, in the NCBI Gene database atwww.ncbi.nlm.nih.gov/gene/3479 (which is incorporated herein byreference as of the date of filing this application).

As used herein, “insulin-like growth factor 1” is used interchangeablywith the term “IGF-1” (and optionally any of the other recognized nameslisted above) refers to the naturally occurring gene that encodes aninsulin-like growth factor 1 protein. The amino acid and complete codingsequences of the reference sequence of the human IGF-1 gene, transcriptvariant 1, mRNA, may be found in, for example, GenBank Accession No. GI:930588898 (RefSeq Accession No. NM_001111283.2; SEQ ID NO: 11; SEQ IDNO: 12); human IGF-1 gene, transcript variant 4, mRNA, may be found atGenBank Accession No. GI: 930616505 (RefSeq Accession No. NM_000618.4;SEQ ID NO: 13 and SEQ ID NO:14); and human IGF-1, transcript variant 2,mRNA, may be found at GenBank Accession No. GI: 163659900 (RefSeqAccession No. NM_001111284.1; SEQ ID NO: 15 and 16. Mammalian orthologsof the human IGF-1 gene may be found in, for example, GI: 930155588(RefSeq Accession No. NM_010512.5, mouseIGF-1; SEQ ID NO:17 and SEQ IDNO:18); GI: 126722710 (RefSeq Accession No. NM_001082478.1, rat; SEQ IDNO:19 and SEQ ID NO:20); GenBank Accession Nos. GI: 544472486 (RefSeqAccession No. XM_005572040.1, cynomolgus monkey; SEQ ID NO:21 and SEQ IDNO:22). Multiple sequence variants for each of the species are known.

A number of naturally occurring SNPs are known and can be found, forexample, in the SNP database at the NCBI atwww.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=3479 (which is incorporatedherein by reference as of the date of filing this application) whichlists SNPs in human IGF-1. In preferred embodiments, such naturallyoccurring variants are included within the scope of the IGF-1 genesequence.

Additional examples of IGF-1 mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an IGFALS gene or an IGF-1gene, including mRNA that is a product ofRNA processing of a primary transcription product. The target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of anIGFALS gene or an IGF-1gene gene. In one embodiment, the target sequenceis within the protein coding region of IGFALS or IGF-1.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. In some embodiments, the target sequence is about19 to about 30 nucleotides in length. In other embodiments, the targetsequence is about 19 to about 25 nucleotides in length. In still otherembodiments, the target sequence is about 19 to about 23 nucleotides inlength. In some embodiments, the target sequence is about 21 to about 23nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine, and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” and “iRNA agent,” “RNA interferenceagent” as used interchangeably herein, refer to an agent that containsRNA as that term is defined herein, and which mediates the targetedcleavage of an RNA transcript via an RNA-induced silencing complex(RISC) pathway. iRNA directs the sequence-specific degradation of mRNAthrough a process known as RNA interference (RNAi). The iRNA modulates,e.g., inhibits, the expression of the target gene, e.g., an IGFALS geneor an IGF-1 gene, in a cell, e.g., a cell within a subject, such as amammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNAi that interacts with a target RNA sequence, e.g., an IGFALSor IGF-1 target mRNA sequence, to direct the cleavage of the target RNA.Without wishing to be bound by theory it is believed that long doublestranded RNA introduced into cells is broken down into double-strandedshort interfering RNAs (siRNAs) comprising a sense strand and anantisense strand by a Type III endonuclease known as Dicer (Sharp et al.(2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme,processes these dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the invention relates to a singlestranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generatedwithin a cell and which promotes the formation of a RISC complex toeffect silencing of the target gene, i.e., an IGFALS or IGF-1 gene.Accordingly, the term “siRNA” is also used herein to refer to an RNAi asdescribed above.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No. 8,101,348and in Lima et al., (2012) Cell 150:883-894, the entire contents of eachof which are hereby incorporated herein by reference. Any of theantisense nucleotide sequences described herein may be used as asingle-stranded siRNA as described herein or as chemically modified bythe methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an IGFALS gene or an IGF-1 gene. Insome embodiments of the invention, a double stranded RNA (dsRNA)triggers the degradation of a target RNA, e.g., an mRNA, through apost-transcriptional gene-silencing mechanism referred to herein as RNAinterference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide or a modified nucleotide. In addition, as used inthis specification, an “iRNA” may include ribonucleotides with chemicalmodifications; an iRNA may include substantial modifications at multiplenucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, or modified nucleobase, or any combinationthereof. Thus, the term modified nucleotide encompasses substitutions,additions or removal of, e.g., a functional group or atom, tointernucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“iRNA” or “RNAi agent” for the purposes of this specification andclaims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments,the hairpin loop can be 10 or fewer nucleotides. In some embodiments,the hairpin loop can be 8 or fewer unpaired nucleotides. In someembodiments, the hairpin loop can be 4-10 unpaired nucleotides. In someembodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs. In one embodiment of theRNAi agent, at least one strand comprises a 3′ overhang of at least 1nucleotide. In another embodiment, at least one strand comprises a 3′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In other embodiments, at least one strandof the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. Incertain embodiments, at least one strand comprises a 5′ overhang of atleast 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or15 nucleotides. In still other embodiments, both the 3′ and the 5′ endof one strand of the RNAi agent comprise an overhang of at least 1nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an IGFALS gene or an IGF-1 gene. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

In some embodiments, an iRNA of the invention is a dsRNA of 24-30nucleotides, or possibly even longer, e.g., 25-35, 27-53, or 27-49nucleotides, that interacts with a target RNA sequence, e.g., an IGFALStarget mRNA sequence or an IGF-1 target mRNA sequence, to direct thecleavage of the target RNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of a doublestranded iRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand, or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end, orboth ends of either an antisense or sense strand of a dsRNA. In oneembodiment of the dsRNA, at least one strand comprises a 3′ overhang ofat least 1 nucleotide. In another embodiment, at least one strandcomprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, atleast one strand of the RNAi agent comprises a 5′ overhang of at least 1nucleotide. In certain embodiments, at least one strand comprises a 5′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′and the 5′ end of one strand of the RNAi agent comprise an overhang ofat least 1 nucleotide.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In certainembodiments, the overhang on the sense strand or the antisense strand,or both, can include extended lengths longer than 10 nucleotides, e.g.,1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15nucleotides in length. In certain embodiments, an extended overhang ison the sense strand of the duplex. In certain embodiments, an extendedoverhang is present on the 3′end of the sense strand of the duplex. Incertain embodiments, an extended overhang is present on the 5′end of thesense strand of the duplex. In certain embodiments, an extended overhangis on the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 3′end of the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the5′end of the antisense strand of the duplex. In certain embodiments, oneor more of the nucleotides in the overhang is replaced with a nucleosidethiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” double stranded RNAi agent is double strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withno nucleotide overhang at one end (i.e., agents with one overhang andone blunt end) or with no nucleotide overhangs at either end.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an IGFALS or IGF-1 mRNA. Asused herein, the term “region of complementarity” refers to the regionon the antisense strand that is substantially complementary to asequence, for example a target sequence, e.g., an IGFALS or IGF-1nucleotide sequence, as defined herein. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches can be in the internal or terminal regions of the molecule.Generally, the most tolerated mismatches are in the terminal regions,e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5′- or 3′-end of theiRNA. In some embodiments, a double stranded RNAi agent of the inventionincludes a nucleotide mismatch in the antisense strand. In someembodiments, a double stranded RNAi agent of the invention includes anucleotide mismatch in the sense strand. In some embodiments, thenucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotidesfrom the 3′-end of the iRNA. In another embodiment, the nucleotidemismatch is, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, “substantially all of the nucleotides are modified” arelargely but not wholly modified and can include not more than 5, 4, 3,2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs or base pairs formedfrom non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a double stranded RNAi agent and a targetsequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding an IGFALS gene or an IGF-1 gene). Forexample, a polynucleotide is complementary to at least a part of anIGFALS or IGF-1 mRNA if the sequence is substantially complementary to anon-interrupted portion of an mRNA encoding an IGFALS or IGF-1 gene.

Accordingly, in some embodiments, the sense strand polynucleotides andthe antisense polynucleotides disclosed herein are fully complementaryto the target IGFALS or IGF-1sequence.

In one embodiment, the antisense polynucleotides disclosed herein arefully complementary to the target IGFALS sequence. In other embodiments,the antisense polynucleotides disclosed herein are substantiallycomplementary to the target IGFALS sequence and comprise a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:1, 3, 5, 7, or 9, or a fragment of any one of SEQ IDNOs:1, 3, 5, 7, or 9, such as about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target IGFALS sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to any one of the sense strand nucleotidesequences in any one of Tables 3, 5, 6, 8, 12, or 14, or a fragment ofany one of the sense strand nucleotide sequences in any one of Tables 3,5, 6, 8, 12, or 14, such as about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target IGFALSsequence and comprises a contiguous nucleotide sequence which is atleast about 80% complementary over its entire length to any one of thesense strand nucleotide sequences in any one of Tables 3, 5, 6, 8, 12,or 14, or a fragment of any one of the sense strand nucleotide sequencesin any one of Tables 3, 5, 6, 8, 12, or 14, such as about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99% complementary.

In one embodiment, the antisense polynucleotides disclosed herein arefully complementary to the target IGF-1 sequence. In other embodiments,the antisense polynucleotides disclosed herein are substantiallycomplementary to the target IGF-1 sequence and comprise a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:11, 13, 15, 17, 19, or 21, or a fragment of any one ofSEQ ID NOs:11, 13, 15, 17, 19, or 21, such as about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target IGF-1 sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to any one of the sense strand nucleotidesequences in any one of Tables 9, 11, 15, 17, 18, or 20, or a fragmentof any one of the sense strand nucleotide sequences in any one of Tables9, 11, 15, 17, 18, or 20, such as about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target IGF-1sequence and comprises a contiguous nucleotide sequence which is atleast about 80% complementary over its entire length to any one of thesense strand nucleotide sequences in any one of Tables 9, 11, 15, 17,18, or 20, or a fragment of any one of the sense strand nucleotidesequences in any one of Tables 9, 11, 15, 17, 18, or 20, such as about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99% complementary.

In an aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisenseoligonucleotide molecule that inhibits a target mRNA via an antisenseinhibition mechanism. The single-stranded antisense oligonucleotidemolecule is complementary to a sequence within the target mRNA. Thesingle-stranded antisense oligonucleotides can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The single-stranded antisense oligonucleotidemolecule may be about 14 to about 30 nucleotides in length and have asequence that is complementary to a target sequence. For example, thesingle-stranded antisense oligonucleotide molecule may comprise asequence that is at least 14, 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from any one of the antisense sequences described herein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as usedherein, includes contacting a cell by any possible means. Contacting acell with an iRNA includes contacting a cell in vitro with the iRNA orcontacting a cell in vivo with the iRNA. The contacting may be donedirectly or indirectly. Thus, for example, the iRNA may be put intophysical contact with the cell by the individual performing the method,or alternatively, the iRNA may be put into a situation that will permitor cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the iRNA. Contacting a cell in vivo may be done, for example,by injecting the iRNA into or near the tissue where the cell is located,or by injecting the iRNA into another area, e.g., the bloodstream or thesubcutaneous space, such that the agent will subsequently reach thetissue where the cell to be contacted is located. For example, the iRNAmay contain or be coupled to a ligand, e.g., GalNAc3, that directs theiRNA to a site of interest, e.g., the liver. Combinations of in vitroand in vivo methods of contacting are also possible. For example, a cellmay also be contacted in vitro with an iRNA and subsequentlytransplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes“introducing” or “delivering the iRNA into the cell” by facilitating oreffecting uptake or absorption into the cell. Absorption or uptake of aniRNA can occur through unaided diffusion or active cellular processes,or by auxiliary agents or devices. Introducing an iRNA into a cell maybe in vitro or in vivo. For example, for in vivo introduction, iRNA canbe injected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and USPublication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or are knownin the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose) that expresses the target gene, either endogenously orheterologously. It is understood that the sequence of the PHD gene mustbe sufficiently complementary to the antisense strand of the iRNA agentfor the agent to be used in the indicated species. In certainembodiments, the subject is a human, such as a human being treated orassessed for a disease, disorder or condition that would benefit fromreduction in an IGFALS gene or an IGF-1 gene expression or replication;a human at risk for a disease, disorder or condition that would benefitfrom reduction in IGFALS or IGF-1 gene expression; a human having adisease, disorder or condition that would benefit from reduction inIGFALS or IGF-1 gene expression; or human being treated for a disease,disorder or condition that would benefit from reduction in IGFALS orIGF-1 gene expression, as described herein. In some embodiments, thesubject is a female human. In other embodiments, the subject is a malehuman.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with IGFALS or IGF-1gene expression or IGFALS or IGF-1 protein production, e.g., acromegaly,cancer. “Treatment” can also mean prolonging survival as compared toexpected survival in the absence of treatment.

The term “lower” or “reduce” in the context of the level of IGFALS orIGF-1 gene expression or IGFALS or IGF-1 protein production in asubject, or a disease marker or symptom refers to a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,or 95%, or below the level of detection for the detection method. Incertain embodiments, the decrease is down to a level accepted as withinthe range of normal for an individual without such disorder which canalso be referred to as a normalization of a level. For example, loweringcholesterol to 180 mg/dl or lower would be considered to be within therange of normal for a subject. A subject having a cholesterol level of230 mg/dl with a cholesterol level decreased to 210 mg/dl would have acholesterol level that was decreased by 40% (230-210/230−180=20/50=40%reduction). In certain embodiments, the reduction is the normalizationof the level of a sign or symptom of a disease, a reduction in thedifference between the subject level of a sign of the disease and thenormal level of the sign for the disease (e.g., the upper level ofnormal when the level must be reduced to reach a normal level, and thelower level of normal when the level must be increased to reach a normallevel). In certain embodiments, the methods include a clinicallyrelevant inhibition of expression of IGFALS or IGF-1, e.g. asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of IGFALS or IGF-1.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an IGFALS gene or an IGF-1 gene or productionof an IGFALS or an IGF-1protein, refers to a reduction in the likelihoodthat a subject will develop a symptom associated with such a disease,disorder, or condition, e.g., a symptom of IGFALS or IGF-1 geneexpression, such as the presence of elevated levels of proteins in theIGF signaling pathway, e.g., acromegaly or cancer. The failure todevelop a disease, disorder or condition, or the reduction in thedevelopment of a symptom or comorbidity associated with such a disease,disorder or condition (e.g., by at least about 10% on a clinicallyaccepted scale for that disease or disorder), or the exhibition ofdelayed symptoms or disease progression (e.g., delayed cancerprogression as determined using RECIST criteria) by days, weeks, monthsor years is considered effective prevention. Prevention may require theadministration of more than one dose.

As used herein, the term “IGF system-associated disease,” usedinterchangeable with the terms “insulin-like growth factor bindingprotein, acid labile subunit-associated disease,” “IGFALS-associateddisease,” “IGF-associated disease,” or “IGF-1-associated disease” is adisease or disorder that is caused by, or associated with IGFALS or IGFgene expression or IGFALS or IGF protein production. The term “IGFsystem-associated disease” includes a disease, disorder or conditionthat would benefit from a decrease in IGFALS or IGF-1 gene expression,replication, or protein activity. Non-limiting examples of IGFsystem-associated diseases include, for example, acromegaly, gigantism,and cancer, especially metastatic cancer.

In certain embodiments, an IGF system-associated disease-associateddisease is acromegaly.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a patient fortreating a subject having acromealgy, cancer, or IGF system-associateddisease, is sufficient to effect treatment of the disease (e.g., bydiminishing, ameliorating or maintaining the existing disease or one ormore symptoms of disease or its related comorbidities). The“therapeutically effective amount” may vary depending on the iRNA, howit is administered, the disease and its severity and the history, age,weight, family history, genetic makeup, stage of pathological processesmediated by IGFALS or IGF-1 gene expression, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated. Treatment may require the administration ofmore than one dose.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subject whodoes not yet experience or display symptoms of acromealgy, cancer, orother IGF system-associated disease-associated diseases, but who may bepredisposed to an IGF system-associated disease-associated disease, issufficient to prevent or delay the development or progression of thedisease or one or more symptoms of the disease for a clinicallysignificant period of time. The “prophylactically effective amount” mayvary depending on the iRNA, how it is administered, the degree of riskof disease, and the history, age, weight, family history, geneticmakeup, the types of preceding or concomitant treatments, if any, andother individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an iRNA that produces some desiredlocal or systemic effect at a reasonable benefit/risk ratio applicableto any treatment. iRNAs employed in the methods of the present inventionmay be administered in a sufficient amount to produce a reasonablebenefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs, or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). A “sample derived from a subject” can refer to blood drawnfrom the subject, or plasma derived therefrom. In certain embodimentswhen detecting a level of IGF-1, a “sample” preferably refers to atissue or body fluid from a subject in which IGF-1 is detectable priorto administration of an agent of the invention, e.g., a liver biopsyfrom a subject with a acromegaly, a tumor. In certain subjects, e.g.,healthy subjects, the level of IGF-1 may not be detectable in a numberof body fluids, cell types, and tissues.

I. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of anIGFALS gene or an IGF-1 gene. In preferred embodiments, the iRNAincludes double stranded ribonucleic acid (dsRNA) molecules forinhibiting the expression of an IGFALS gene or an IGF-1 gene in a cell,such as a cell within a subject, e.g., a mammal, such as a human havingan IGF system-associated disease-associated disease, e.g., acromeagly.The dsRNAi agent includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an IGFALS gene or an IGF-1 gene. The regionof complementarity is about 30 nucleotides or less in length (e.g.,about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotidesor less in length). Upon contact with a cell expressing the IGFALS geneor the IGF-1 gene, the iRNA inhibits the expression of the IGFALS geneor the IGF-1 gene (e.g., a human, a primate, a non-primate, or a birdIGFALS gene or IGF-1 gene) by at least 20%, preferably at least 30%, asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, western blotting or flowcytometric techniques. Inpreferred embodiments, inhibition of expression is determined by theqPCR method provided in the examples. For in vitro assessment ofactivity, percent inhibition is determined using the methods provided inExample 2 at a single dose at a 10 nM duplex final concentration. For invivo studies, the level after treatment can be compared to, for example,an appropriate historical control or a pooled population sample controlto determine the level of reduction, e.g., when a baseline value is noavailable for the subject.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an IGFALSgene or IGF-1 gene. The other strand (the sense strand) includes aregion that is complementary to the antisense strand, such that the twostrands hybridize and form a duplex structure when combined undersuitable conditions. As described elsewhere herein and as known in theart, the complementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is about 15 to 30 base pairs in length,e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges andlengths intermediate to the above recited ranges and lengths are alsocontemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is about15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

In some embodiments, the dsRNA is about 15 to 23 nucleotides in length,or about 25 to 30 nucleotides in length. In general, the dsRNA is longenough to serve as a substrate for the Dicer enzyme. For example, it iswell-known in the art that dsRNAs longer than about 21-23 nucleotides inlength may serve as substrates for Dicer. As the ordinarily skilledperson will also recognize, the region of an RNA targeted for cleavagewill most often be part of a larger RNA molecule, often an mRNAmolecule. Where relevant, a “part” of an mRNA target is a contiguoussequence of an mRNA target of sufficient length to allow it to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to about 36 base pairs, e.g., 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target IGFALS or IGF-1 gene expression is not generated in the targetcell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3,or 4 nucleotides. dsRNAs having at least one nucleotide overhang canhave superior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand, or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of an antisense or sensestrand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

Double stranded RNAi compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double strandedRNA molecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Similarly, single-stranded oligonucleotides of the invention can beprepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention for inhibing the expression of anIGFALS gene includes at least two nucleotide sequences, a sense sequenceand an anti-sense sequence. The sense strand is selected from the groupof sequences provided in any one of Tables 3, 5, 6, 8, 12, and 14, andthe corresponding antisense strand of the sense strand is selected fromthe group of sequences in any one of Tables 3, 5, 6, 8, 12, and 14. Inthis aspect, one of the two sequences is complementary to the other ofthe two sequences, with one of the sequences being substantiallycomplementary to a sequence of an mRNA generated in the expression of anIGFALS gene. As such, in this aspect, a dsRNA will include twooligonucleotides, where one oligonucleotide is described as the sensestrand in any one of Table 3, 5, 6, 8, 12, and 14, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Table 3, 5, 6, 8, 12, and 14. In certainembodiments, the substantially complementary sequences of the dsRNA arecontained on separate oligonucleotides. In other embodiments, thesubstantially complementary sequences of the dsRNA are contained on asingle oligonucleotide.

In an aspect, a dsRNA of the invention for inhibing the expression of anIGF-1 gene includes at least two nucleotide sequences, a sense sequenceand an anti-sense sequence. The sense strand is selected from the groupof sequences provided in any one of Tables 9, 11, 15, 17, 18, and 20,and the corresponding antisense strand of the sense strand is selectedfrom the group of sequences in any one of Tables 9, 11, 15, 17, 18, and20. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of an IGF-1 gene. As such, in this aspect, a dsRNA willinclude two oligonucleotides, where one oligonucleotide is described asthe sense strand in any one of Table 9, 11, 15, 17, 18, and 20, and thesecond oligonucleotide is described as the corresponding antisensestrand of the sense strand in any one of Table 9, 11, 15, 17, 18, and20. In certain embodiments, the substantially complementary sequences ofthe dsRNA are contained on separate oligonucleotides. In otherembodiments, the substantially complementary sequences of the dsRNA arecontained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 3, 6, 9,12, 15, and 18 are not described as modified or conjugated sequences,the RNA of the iRNA of the invention e.g., a dsRNA of the invention, maycomprise any one of the sequences set forth in any one of Tables 3, 6,9, 12, 15, and 18, or the sequences of any one of Tables 5, 8, 11, 14,17, and 20 that are modified, or the sequences of any one of Tables 5,8, 11, 14, 17, and 20 that are conjugated to a ligand. In other words,the invention encompasses dsRNAs of any one of Tables 3, 5, 6, 8, 9, 11,12, 14, 15, 17, 18, and 20 which are un-modified, un-conjugated,modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3, 5, 6, 8, 9,11, 12, 14, 15, 17, 18, and 20, dsRNAs described herein can include atleast one strand of a length of minimally 21 nucleotides. It can bereasonably expected that shorter duplexes having one of the sequences ofTables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20 minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences of Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17,18, and 20, and differing in their ability to inhibit the expression ofan IGFALS gene or an IGF-1 gene by not more than about 5, 10, 15, 20,25, or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15,17, 18, and 20 identify a site(s) in an IGFALS transcript or IGF-1transcript that is susceptible to RISC-mediated cleavage. As such, thepresent invention further features iRNAs that target within one of thesesites. As used herein, an iRNA is said to target within a particularsite of an RNA transcript if the iRNA promotes cleavage of thetranscript anywhere within that particular site. Such an iRNA willgenerally include at least about 15 contiguous nucleotides from one ofthe sequences provided in Tables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18,and 20 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an IGFALS gene or an IGF-1 gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art or provided herein) to identify thosesequences that perform optimally can identify those RNA sequences that,when targeted with an iRNA agent, mediate the best inhibition of targetgene expression. Thus, while the sequences identified, for example, inTables 3 and 5 represent effective target sequences, it is contemplatedthat further optimization of inhibition efficiency can be achieved byprogressively “walking the window” one nucleotide upstream or downstreamof the given sequences to identify sequences with equal or betterinhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, and 20, furtheroptimization could be achieved by systematically either adding orremoving nucleotides to generate longer or shorter sequences and testingthose sequences generated by walking a window of the longer or shortersize up or down the target RNA from that point. Again, coupling thisapproach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an IGFALS gene or an IGF-1 gene,generally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of an IGFALSgene or IGF-1 gene. Consideration of the efficacy of iRNAs withmismatches in inhibiting expression of an IGFALS gene or an IGF-1 geneis important, especially if the particular region of complementarity inan IGFALS gene or an IGF-1 gene is known to have polymorphic sequencevariation within the population.

II. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., adsRNA, is unmodified, and does not comprise, e.g., chemicalmodifications or conjugations known in the art and described herein. Inother embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA,is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA orsubstantially all of the nucleotides of an iRNA are modified, i.e., notmore than 5, 4, 3, 2, or 1 unmodified nucleotides are present in astrand of the iRNA.

The nucleic acids featured in the invention can be synthesized ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; or backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative US patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts.

Representative US patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein,in which both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with novel groups. Thebase units are maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound in which an RNAmimetic that has been shown to have excellent hybridization propertiesis referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative US patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents ofeach of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position:OH; F; O-, S-, or N-alkyl; O, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O-CH₂-O-CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative US patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative US patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH₂-O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′(see, e.g., US Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative US patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and -C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

Representative US publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT (idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an iRNA.Suitable phosphate mimics are disclosed in, for example US PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO2013/075035, the entire contents of each of which areincorporated herein by reference. WO2013/075035 provides motifs of threeidentical modifications on three consecutive nucleotides into a sensestrand or antisense strand of a dsRNAi agent, particularly at or nearthe cleavage site. In some embodiments, the sense strand and antisensestrand of the dsRNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense or antisense strand. The dsRNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand.

More specifically, when the sense strand and antisense strand of thedouble stranded RNAi agent are completely modified to have one or moremotifs of three identical modifications on three consecutive nucleotidesat or near the cleavage site of at least one strand of a dsRNAi agent,the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., IGFALS or IGF-1gene) in vivo. The RNAi agent comprises a sense strand and an antisensestrand. Each strand of the RNAi agent may be, independently, 12-30nucleotides in length. For example, each strand may independently be14-30 nucleotides in length, 17-30 nucleotides in length, 25-30nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides inlength, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides inlength, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” Theduplex region of a dsRNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be 14-30 nucleotide pairs in length,17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length,17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length,17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length,19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length,21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.In another example, the duplex region is selected from 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In certain embodiments, the sense and antisense strands may be evenlonger. For example, in certain embodiments, the sense strand and theantisense strand are independently 25-35 nucleotides in length. Incertain embodiments, each the sense and the antisense strand areindependently 27-53 nucleotides in length, e.g., 27-49, 31-49, 33-49,35-49, 37-49, and 39-49 nucleotides in length. In certain embodiments,the dsRNAi agent may contain one or more overhang regions or cappinggroups at the 3′-end, 5′-end, or both ends of one or both strands. Theoverhang can be, independently, 1-6 nucleotides in length, for instance2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides inlength, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides inlength. In certain embodiments, at least one strand of the dsRNAi agentcomprises a 3′ overhang of at least 1 nucleotide. In another embodiment,at least one strand comprises a 3′ overhang of at least 2 nucleotides,e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. Inother embodiments, at least one strand of the dsRNAi agent comprises a5′ overhang of at least 1 nucleotide. In certain embodiments, at leastone strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2,3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still otherembodiments, both the 3′ and the 5′ end of one strand of the dsRNAiagent comprise an overhang of at least 1 nucleotide.

In certain embodiments, the overhang regions can include extendedoverhang regions as provided above. The overhangs can be the result ofone strand being longer than the other, or the result of two strands ofthe same length being staggered. The overhang can form a mismatch withthe target mRNA or it can be complementary to the gene sequences beingtargeted or can be another sequence. The first and second strands canalso be joined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In certain embodiments, the nucleotides in the overhang region of thedsRNAi agent can each independently be a modified or unmodifiednucleotide including, but no limited to 2′-sugar modified, such as,2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine(Teo), 2′-O-methoxyethyladenosine (Aeo),2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof. For example, TT can be an overhang sequence for either end oneither strand. The overhang can form a mismatch with the target mRNA orit can be complementary to the gene sequences being targeted or can beanother sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or bothstrands of the dsRNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In some embodiments, the overhang ispresent at the 3′-end of the sense strand, antisense strand, or bothstrands. In some embodiments, this 3′-overhang is present in theantisense strand. In some embodiments, this 3′-overhang is present inthe sense strand.

The dsRNAi agent may contain only a single overhang, which canstrengthen the interference activity of the RNAi, without affecting itsoverall stability. For example, the single-stranded overhang may belocated at the 3′-end of the sense strand or, alternatively, at the3′-end of the antisense strand. The RNAi may also have a blunt end,located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of thedsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. While not wishing to be bound by theory, the asymmetric blunt endat the 5′-end of the antisense strand and 3′-end overhang of theantisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of19 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sensestrand and a 23 nucleotide antisense strand, wherein the sense strandcontains at least one motif of three 2′-F modifications on threeconsecutive nucleotides at positions 9, 10, 11 from the 5′end; theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at positions 11, 12, 13from the 5′end, wherein one end of the RNAi agent is blunt, while theother end comprises a 2 nucleotide overhang. Preferably, the 2nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In certain embodiments, everynucleotide in the sense strand and the antisense strand of the dsRNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In certain embodiments each residue isindependently modified with a 2′-O-methyl or 3′-fluoro, e.g., in analternating motif. Optionally, the dsRNAi agent further comprises aligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and anantisense strand, wherein the sense strand is 25-30 nucleotide residuesin length, wherein starting from the 5′ terminal nucleotide (position 1)positions 1 to 23 of the first strand comprise at least 8ribonucleotides; the antisense strand is 36-66 nucleotide residues inlength and, starting from the 3′ terminal nucleotide, comprises at least8 ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In certain embodiments, the dsRNAi agent comprises sense and antisensestrands, wherein the dsRNAi agent comprises a first strand having alength which is at least 25 and at most 29 nucleotides and a secondstrand having a length which is at most 30 nucleotides with at least onemotif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ endof the first strand and the 5′ end of the second strand form a blunt endand the second strand is 1-4 nucleotides longer at its 3′ end than thefirst strand, wherein the duplex region region which is at least 25nucleotides in length, and the second strand is sufficientlycomplemenatary to a target mRNA along at least 19 nucleotide of thesecond strand length to reduce target gene expression when the RNAiagent is introduced into a mammalian cell, and wherein Dicer cleavage ofthe dsRNAi agent preferentially results in an siRNA comprising the3′-end of the second strand, thereby reducing expression of the targetgene in the mammal. Optionally, the dsRNAi agent further comprises aligand.

In certain embodiments, the sense strand of the dsRNAi agent contains atleast one motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In certain embodiments, the antisense strand of the dsRNAi agent canalso contain at least one motif of three identical modifications onthree consecutive nucleotides, where one of the motifs occurs at or nearthe cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 17-23 nucleotides inlength, the cleavage site of the antisense strand is typically aroundthe 10, 11, and 12 positions from the 5′-end. Thus the motifs of threeidentical modifications may occur at the 9, 10, 11 positions; the 10,11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; orthe 13, 14, 15 positions of the antisense strand, the count startingfrom the first nucleotide from the 5′-end of the antisense strand, or,the count starting from the first paired nucleotide within the duplexregion from the 5′-end of the antisense strand. The cleavage site in theantisense strand may also change according to the length of the duplexregion of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may containmore than one motif of three identical modifications on threeconsecutive nucleotides. The first motif may occur at or near thecleavage site of the strand and the other motifs may be a wingmodification. The term “wing modification” herein refers to a motifoccurring at another portion of the strand that is separated from themotif at or near the cleavage site of the same strand. The wingmodification is either adjacent to the first motif or is separated by atleast one or more nucleotides. When the motifs are immediately adjacentto each other then the chemistries of the motifs are distinct from eachother, and when the motifs are separated by one or more nucleotide thanthe chemistries can be the same or different. Two or more wingmodifications may be present. For instance, when two wing modificationsare present, each wing modification may occur at one end relative to thefirst motif which is at or near cleavage site or on either side of thelead motif.

Like the sense strand, the antisense strand of the dsRNAi agent maycontain more than one motif of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two terminal nucleotides at the 3′-end, 5′-end, or bothends of the strand.

In other embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two paired nucleotides within the duplex region at the3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two,or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two, or three nucleotides; two modifications each from one strand fallon the other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In some embodiments, every nucleotide in the sense strand and antisensestrand of the dsRNAi agent, including the nucleotides that are part ofthe motifs, may be modified. Each nucleotide may be modified with thesame or different modification which can include one or more alterationof one or both of the non-linking phosphate oxygens or of one or more ofthe linking phosphate oxygens; alteration of a constituent of the ribosesugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′- or 5′-terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a dsRNAi agent or may only occur in a single strandregion of a dsRNAi agent. For example, a phosphorothioate modificationat a non-linking O position may only occur at one or both ends, may onlyoccur in a terminal region, e.g., at a position on a terminalnucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, ormay occur in double strand and single strand regions, particularly atthe ends. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang,or in both. For example, it can be desirable to include purinenucleotides in overhangs. In some embodiments all or some of the basesin a 3′- or 5′-overhang may be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ position of the ribose sugar with modificationsthat are known in the art, e.g., the use of deoxyribonucleotides,2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of theribosugar of the nucleobase, and modifications in the phosphate group,e.g., phosphorothioate modifications. Overhangs need not be homologouswith the target sequence.

In some embodiments, each residue of the sense strand and antisensestrand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In some embodiments, the dsRNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′ to 3′ of the strand and the alternating motif inthe antisense strand may start with “BABABA” from 5′ to 3′ of the strandwithin the duplex region. As another example, the alternating motif inthe sense strand may start with “AABBAABB” from 5′ to 3′ of the strandand the alternating motif in the antisense strand may start with“BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so thatthere is a complete or partial shift of the modification patternsbetween the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand or antisense strandinterrupts the initial modification pattern present in the sense strandor antisense strand. This interruption of the modification pattern ofthe sense or antisense strand by introducing one or more motifs of threeidentical modifications on three consecutive nucleotides to the sense orantisense strand may enhance the gene silencing activity against thetarget gene.

In some embodiments, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)or N_(b) may be present or absent when there is a wing modificationpresent.

The iRNA may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand, antisense strand, or both strands in anyposition of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand or antisense strand; or thesense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8phosphorothioateinternucleotide linkages. In some embodiments, the antisense strandcomprises two phosphorothioate internucleotide linkages at the 5′-endand two phosphorothioate internucleotide linkages at the 3′-end, and thesense strand comprises at least two phosphorothioate internucleotidelinkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, or the 5′end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, thedsRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mistmatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of thefirst 1, 2, 3, 4, or 5 base pairs within the duplex regions from the5′-end of the antisense strand independently selected from the group of:A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other thancanonical pairings or pairings which include a universal base, topromote the dissociation of the antisense strand at the 5′-end of theduplex.

In certain embodiments, the nucleotide at the 1 position within theduplex region from the 5′-end in the antisense strand is selected fromA, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or3 base pair within the duplex region from the 5′-end of the antisensestrand is an AU base pair. For example, the first base pair within theduplex region from the 5′-end of the antisense strand is an AU basepair.

In other embodiments, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisensestrand is deoxy-thymine (dT). For example, there is a short sequence ofdeoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-endof the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented byformula (I):5′n_(p)-N_(a)-(XXX)_(i)-N_(b)- YYY- N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications ofalternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage siteof the sense strand. For example, when the dsRNAi agent has a duplexregion of 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7,8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sensestrand, the count starting from the first nucleotide, from the 5′-end;or optionally, the count starting at the first paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Ib);5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′  (Ic); or5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2, or 0 modified nucleotides. Each N_(a) can independently representan oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_(b) is 0,1, 2, 3, 4, 5, or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):5′n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(n)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNAi agent has a duplex region of 17-23nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10,11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the first nucleotide, from the5′-end; or optionally, the count starting at the first paired nucleotidewithin the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motifoccurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or bothk and l are 1.

The antisense strand can therefore be represented by the followingformulas:5′n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p′)3′  (IIb);5′n_(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′)3′  (IIc); or5′n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIC), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5, or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:5′n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be thesame or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may containYYY motif occurring at 9, 10, and 11 positions of the strand when theduplex region is 21 nt, the count starting from the first nucleotidefrom the 5′-end, or optionally, the count starting at the first pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe first nucleotide from the 5′-end, or optionally, the count startingat the first paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the dsRNAi agents for use in the methods of the inventionmay comprise a sense strand and an antisense strand, each strand having14 to 30 nucleotides, the iRNA duplex represented by formula (III):sense: 5′n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q)3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand formingan iRNA duplex include the formulas below:5′n_(p)-N_(a)-YYY-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′5′   (IIIa)5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′5′   (IIIb)5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′5′   (IIIc)5′n_(p)-N_(a)-XXX-N_(b)′-YYY-N_(b)-ZZZ-N_(a)-n_(q)3′3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′5′  (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a), N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b), and N_(b) independently comprises modifications ofalternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and(IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide isdifferent than the modification on the Y′ nucleotide, the modificationon the Z nucleotide is different than the modification on the Z′nucleotide, and/or the modification on the X nucleotide is differentthan the modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications. In other embodiments, when the RNAi agent is representedby formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet otherembodiments, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a monovalent, a bivalent or atrivalent branched linker (described below). In other embodiments, whenthe RNAi agent is represented by formula (IIId), the N_(a) modificationsare 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least onen_(p)′ is linked to a neighboring nucleotide via phosphorothioatelinkage, the sense strand comprises at least one phosphorothioatelinkage, and the sense strand is conjugated to one or more GalNAcderivatives attached through a monovalent, a bivalent or a trivalentbranched linker.

In some embodiments, when the dsRNAi agent is represented by formula(IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications, n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via phosphorothioate linkage, the sense strandcomprises at least one phosphorothioate linkage, and the sense strand isconjugated to one or more GalNAc derivatives attached through amonovalent, a bivalent or a trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at leasttwo duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In some embodiments, the dsRNAi agent is a multimer containing three,four, five, six, or more duplexes represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId), wherein the duplexes are connected by alinker. The linker can be cleavable or non-cleavable. Optionally, themultimer further comprises a ligand. Each of the duplexes can target thesame gene or two different genes; or each of the duplexes can targetsame gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one offormulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to eachother at the 5′ end, and one or both of the 3′ ends, and are optionallyconjugated to a ligand. Each of the agents can target the same gene ortwo different genes; or each of the agents can target same gene at twodifferent target sites.

Various publications describe multimeric iRNAs that can be used in themethods of the invention. Such publications include U.S. Pat. No.7,858,769, WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887,and WO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the iRNA that contains conjugationsof one or more carbohydrate moieties to an iRNA can optimize one or moreproperties of the iRNA. In many cases, the carbohydrate moiety will beattached to a modified subunit of the iRNA. For example, the ribosesugar of one or more ribonucleotide subunits of a iRNA can be replacedwith another moiety, e.g., a non-carbohydrate (preferably cyclic)carrier to which is attached a carbohydrate ligand. A ribonucleotidesubunit in which the ribose sugar of the subunit has been so replaced isreferred to herein as a ribose replacement modification subunit (RRMS).A cyclic carrier may be a carbocyclic ring system, i.e., all ring atomsare carbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, anddecalin; preferably, the acyclic group is a serinol backbone ordiethanolamine backbone.

In certain embodiments, the iRNA for use in the methods of the inventionfor inhibiting the expression of an IGFALS gene is an agent selectedfrom the agents listed in any one of Tables 3, 5, 6, 8, 12, and 14.These agents may further comprise a ligand. These agents may furthercomprise a ligand.

In certain embodiments, the iRNA for use in the methods of the inventionfor inhibiting the expression of an IGF-1 gene is an agent selected fromthe agents listed in any one of Tables 9, 11, 15, 17, 18, and 20. Theseagents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the iRNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the iRNA e.g., into a cell. For example, the ligand can beattached to the sense strand, antisense strand or both strands, at the3′-end, 5′-end or both ends. For instance, the ligand may be conjugatedto the sense strand. In preferred embodiments, the ligand is conjugatedto the 3′-end of the sense strand. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid(Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060). Incertain embodiments, the modification can include a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands do nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, monovalent or multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate,polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidemimetic. In certain embodiments, ligands include monovalent ormultivalent galactose. In certain embodiments, ligands includecholesterol.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, or intermediate filaments. The drug can be, for example,taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases, or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the useof an oligonucleotide that bears a pendant reactive functionality, suchas that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive oligonucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates In certain embodiments, the ligand or conjugate is alipid or lipid-based molecule. Such a lipid or lipid-based moleculepreferably binds a serum protein, e.g., human serum albumin (HSA). AnHSA binding ligand allows for distribution of the conjugate to a targettissue, e.g., a non-kidney target tissue of the body. For example, thetarget tissue can be the liver, including parenchymal cells of theliver. Other molecules that can bind HSA can also be used as ligands.For example, naproxen or aspirin can be used. A lipid or lipid-basedligand can (a) increase resistance to degradation of the conjugate, (b)increase targeting or transport into a target cell or cell membrane, or(c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, itbinds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In other embodiments, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:24). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:25) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:26) and theDrosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:27) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA further comprises a carbohydrate. The carbohydrate conjugated iRNAis advantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which can be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which can be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri-, and oligosaccharides containingfrom about 4, 5, 6, 7, 8, or 9 monosaccharide units), andpolysaccharides such as starches, glycogen, cellulose and polysaccharidegums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7,or C8) sugars; di- and trisaccharides include sugars having two or threemonosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in thecompositions and methods of the invention is a monosaccharide.

In other embodiments, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the group:

In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc)or GalNAc derivative. In certain embodiments of the invention, theGalNAc or GalNAc derivative is attached to an iRNA agent of theinvention via a monovalent linker. In some embodiments, the GalNAc orGalNAc derivative is attached to an iRNA agent of the invention via abivalent linker. In yet other embodiments of the invention, the GalNAcor GalNAc derivative is attached to an iRNA agent of the invention via atrivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Inanother embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

The GalNAc or GalNAc derivative may be conjugated to the 3′ end of thesense strand of the double stranded RNAi agent, the 5′ end of the sensestrand of the double stranded RNAi agent, the 3′ end of the antisensestrand of the double stranded RNAi agent, or the 5′ end of the antisensestrand of the double stranded RNAi agent. In certain embodiments, themonosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the presentinvention include those described in PCT Publication Nos. WO 2014/179620and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17,6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, or 100 times faster in a target cell or under a first referencecondition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential, or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100times faster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood or serum (or under invitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(-S-S-). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-,-S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-, P(S)(ORk)-S-,-S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-,-S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. Preferred embodimentsare -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-, P(S)(SH)-O-, -S-P(O)(OH)-O-,-O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-,-O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-,-S-P(O)(H)-S-, and -O-P(S)(H)-S-. A preferred embodiment is-O-P(O)(OH)-O-. These candidates can be evaluated using methodsanalogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or byagents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula -C═NN-, C(O)O, or -OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include, but are not limited to,esters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula -C(O)O-, or -OC(O)-. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a monovalent, a bivalent or a trivalent branchedlinker.

In certain embodiments, a dsRNA of the invention is conjugated to amonovalent, a bivalent or a trivalent branched linker selected from thegroup of structures shown in any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH, or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C, or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), -C(O)—CH(R^(a))-NH-, CO, CH═N-O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B), andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable monovalent, bivalent and trivalent branched linkergroups conjugating GalNAc derivatives include, but are not limited to,the structures recited above as formulas II, VII, XI, X, and XIII.

Representative US patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; and 8,106,022, the entire contents of each ofwhich are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAi agents, that containtwo or more chemically distinct regions, each made up of at least onemonomer unit, i.e., a nucleotide in the case of a dsRNA compound. TheseiRNAs typically contain at least one region wherein the RNA is modifiedso as to confer upon the iRNA increased resistance to nucleasedegradation, increased cellular uptake, or increased binding affinityfor the target nucleic acid. An additional region of the iRNA can serveas a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of iRNA inhibition of gene expression.Consequently, comparable results can often be obtained with shorteriRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof RNAs bearing an aminolinker at one or more positions of the sequence.The amino group is then reacted with the molecule being conjugated usingappropriate coupling or activating reagents. The conjugation reactioncan be performed either with the RNA still bound to the solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disease, disorder, or condition associated withIGFALS gene or IGF-1 gene expression) can be achieved in a number ofdifferent ways. For example, delivery may be performed by contacting acell with an iRNA of the invention either in vitro or in vivo. In vivodelivery may also be performed directly by administering a compositioncomprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivodelivery may be performed indirectly by administering one or morevectors that encode and direct the expression of the iRNA. Thesealternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when a dsRNAi agent isadministered locally. For example, intraocular delivery of a VEGF dsRNAby intravitreal injection in cynomolgus monkeys (Tolentino, M J, et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA to thetarget tissue and avoid undesirable off-target effects. iRNA moleculescan be modified by chemical conjugation to lipophilic groups such ascholesterol to enhance cellular uptake and prevent degradation. Forexample, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O, et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H, et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R, et al(2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N, et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal,A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A.(2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu,S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, etal (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm.Res. 16:1799-1804). In some embodiments, an iRNA forms a complex withcyclodextrin for systemic administration. Methods for administration andpharmaceutical compositions of iRNAs and cyclodextrins can be found inU.S. Pat. No. 7,427,605, which is herein incorporated by reference inits entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting an IGFALS gene or an IGF-1 gene can be expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an IGFALS gene or an IGF-1 gene. Such pharmaceuticalcompositions are formulated based on the mode of delivery. One exampleis compositions that are formulated for systemic administration viaparenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), orintravenous (IV) delivery.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an IGFALS gene or an IGF-1gene. In general, a suitable dose of an iRNA of the invention will be inthe range of about 0.001 to about 200.0 milligrams per kilogram bodyweight of the recipient per day, generally in the range of about 1 to 50mg per kilogram body weight per day. Typically, a suitable dose of aniRNA of the invention will be in the range of about 0.1 mg/kg to about5.0 mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-doseregimen may include administration of a therapeutic amount of iRNA on aregular basis, such as every other day or once a year. In certainembodiments, the iRNA is administered about once per month to about onceper quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration weekly orbiweekly for three months, administration can be repeated once permonth, for six months, or a year; or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a composition can include a single treatment or aseries of treatments. Estimates of effective dosages and in vivohalf-lives for the individual iRNAs encompassed by the invention can bemade using conventional methodologies or on the basis of in vivo testingusing an appropriate animal model, as known in the art. For example, amouse model of acromegaly was developed by Kovacs et al. (1997,Endocrinology) the entire contents of which are incorporated herein byreference. Bovine growth hormone transgenic mice also exhibit featuresof acromegaly (Palmiter et al., Science (1983), Olsson et al., Am J PhysEndo Metab (2003), Berryman et al, GH and IGF Res (2004), Izzard et al.,GH and IGF Res (2009), Blutke et al., Mol and Cell Endo (2014)).Multiple animal models of cancer are known in the art.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal, or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricularadministration.

The iRNA can be delivered in a manner to target a particular tissue(e.g., liver cells).

Pharmaceutical compositions and formulations for topical or transdermaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable. Coated condoms,gloves and the like can also be useful. Suitable topical formulationsinclude those in which the iRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). iRNAs featured in the invention can be encapsulatedwithin liposomes or can form complexes thereto, in particular tocationic liposomes. Alternatively, iRNAs can be complexed to lipids, inparticular to cationic lipids. Suitable fatty acids and esters includebut are not limited to arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA. The lipophilic material isolates the aqueous interiorfrom an aqueous exterior, which typically does not include the iRNAcomposition, although in some examples, it may. Liposomes are useful forthe transfer and delivery of active ingredients to the site of action.Because the liposomal membrane is structurally similar to biologicalmembranes, when liposomes are applied to a tissue, the liposomal bilayerfuses with bilayer of the cellular membranes. As the merging of theliposome and cell progresses, the internal aqueous contents that includethe iRNA are delivered into the cell where the iRNA can specificallybind to a target RNA and can mediate RNA interference. In some cases theliposomes are also specifically targeted, e.g., to direct the iRNA toparticular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of two or more of phospholipid, phosphatidylcholine, andcholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185 and 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In some embodiments, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg,Md.) is an effective agent for the delivery of highly anionic nucleicacids into living tissue culture cells that comprise positively chargedDOTMA liposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNAs can bedelivered, for example, subcutaneously by infection in order to deliveriRNAs to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inWO/2008/042973.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of iRNA, analkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds.Exemplary micelle forming compounds include lecithin, hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid, glycolic acid,lactic acid, chamomile extract, cucumber extract, oleic acid, linoleicacid, linolenic acid, monoolein, monooleates, monolaurates, borage oil,evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine andpharmaceutically acceptable salts thereof, glycerin, polyglycerin,lysine, polylysine, triolein, polyoxyethylene ethers and analoguesthereof, polidocanol alkyl ethers and analogues thereof,chenodeoxycholate, deoxycholate, and mixtures thereof. The micelleforming compounds may be added at the same time or after addition of thealkali metal alkyl sulphate. Mixed micelles will form with substantiallyany kind of mixing of the ingredients but vigorous mixing in order toprovide smaller size micelles.

In one method a first micellar composition is prepared which containsthe RNAi and at least the alkali metal alkyl sulphate. The firstmicellar composition is then mixed with at least three micelle formingcompounds to form a mixed micellar composition. In another method, themicellar composition is prepared by mixing the RNAi, the alkali metalalkyl sulphate and at least one of the micelle forming compounds,followed by addition of the remaining micelle forming compounds, withvigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol or m-cresol may be added with the micelle formingingredients. An isotonic agent such as glycerin may also be added afterformation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAi agents of the invention may be fully encapsulated ina lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; USPublication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In some embodiments, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles.

In some embodiments, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see US20090023673,which is incorporated herein by reference), Cholesterol (Sigma-Aldrich),and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 Exemplary lipid formulations cationic lipid/non-cationiclipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic LipidLipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA)(57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)—N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG- DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which is herebyincorporated by reference.

MC3 comprising formulations are described, e.g., in US PatentPublication No. 2010/0324120, filed Jun. 10, 2010, the entire contentsof which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in WO2010/129709, which ishereby incorporated by reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions, or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acids oresters or salts thereof, bile acids or salts thereof. Suitable bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG), and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses, and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular, or intrahepatic administrationcan include sterile aqueous solutions which can also contain buffers,diluents, and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds, and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids, and self-emulsifying semisolids. Formulationsinclude those that target the liver when treating hepatic disorders suchas hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The iRNAs of the present invention can be prepared and formulated asemulsions. Emulsions are typically heterogeneous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution either in the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin, and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate, and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andantioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, andparenteral routes, and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the iRNAs are formulated asmicroemulsions. A microemulsion can be defined as a system of water,oil, and amphiphile which is a single optically isotropic andthermodynamically stable liquid solution (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij® 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex® 300, Captex® 355,Capmul® MCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils, and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill® 3),Labrasol®, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories-surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA and (b) one or more agents whichfunction by a non-iRNA mechanism and which are useful in treating anIGFALS or IGF-1-associated disorder.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby IGFALS or IGF-1 expression. In any event, the administering physiciancan adjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VI. Methods of the Invention

The present invention also provides methods of inhibiting expression ofan IGFALS gene or IGF-1 gene in a cell. The methods include contacting acell with an RNAi agent, e.g., double stranded RNAi agent, in an amounteffective to inhibit expression of IGFALS or IGF-1 in the cell, therebyinhibiting expression of IGFALS or IGF-1 in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNAi agent,may be done in vitro or in vivo. Contacting a cell in vivo with the iRNAincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the iRNA. Combinations of in vitro and in vivomethods of contacting a cell are also possible. Contacting a cell may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In preferred embodiments, thetargeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, orany other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an IGFALS or “inhibiting expressionof an IGF-1” is intended to refer to inhibition of expression of anyIGFALS gene or IGF-1 gene (such as, e.g., a mouse IGFALS gene or IGF-1gene, a rat IGFALS gene or IGF-1 gene, a monkey IGFALS gene or IGF-1gene, or a human IGFALS gene or IGF-1 gene) as well as variants ormutants of an IGFALS gene or IGF-1 gene. Thus, the IGFALS gene or IGF-1gene may be a wild-type IGFALS gene or IGF-1 gene, a mutant IGFALS geneor IGF-1 gene (such as a mutant IGFALS gene or IGF-1 gene), or atransgenic IGFALS gene or IGF-1 gene in the context of a geneticallymanipulated cell, group of cells, or organism.

“Inhibiting expression of an IGFALS gene” or “inhibiting expression ofan IGF-1 gene” includes any level of inhibition of an IGFALS gene or anIGF-1 gene, e.g., at least partial suppression of the expression of anIGFALS gene or an IGF-1 gene. The expression of the IGFALS gene or anIGF-1 genemay be assessed based on the level, or the change in thelevel, of any variable associated with IGFALS gene or an IGF-1geneexpression, e.g., IGFALS mRNA or IGF-1 mRNA level or an IGFALSprotein level or an IGF-1 protein level. This level may be assessed inan individual cell or in a group of cells, including, for example, asample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with IGFALS or IGF-1expression compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of anIGFALS or IGF-1 gene is inhibited by at least 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level ofdetection of the assay. In some embodiments, the inhibition ofexpression of an IGFALS gene or an IGF-1 gene results in normalizationof the level of IGF-1 such that the difference between the level beforetreatment and a normal control level is reduced by at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Inhibition of the expression of an IGFALS gene or an IGF-1 gene may bemanifested by a reduction of the amount of mRNA expressed by a firstcell or group of cells (such cells may be present, for example, in asample derived from a subject) in which an IGFALS gene or an IGF-1 geneis transcribed and which has or have been treated (e.g., by contactingthe cell or cells with an iRNA of the invention, or by administering aniRNA of the invention to a subject in which the cells are or werepresent) such that the expression of an IGFALS gene or an IGF-1 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has not or havenot been so treated (control cell(s) not treated with an iRNA or nottreated with an iRNA targeted to the gene of interest). In preferredembodiments, the inhibition is assessed by the method provided inExample 2 and expressing the level of mRNA in treated cells as apercentage of the level of mRNA in control cells, using the followingformula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{11mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of an IGFALS gene oran IGF-1 gene may be assessed in terms of a reduction of a parameterthat is functionally linked to IGFALS or IGF-1 gene expression, e.g.,IGFALS or IGF-1 protein expression or IGF signaling pathways. IGFALS orIGF-1 gene silencing may be determined in any cell expressing IGFALS orIGF-1, either endogenous or heterologous from an expression construct,and by any assay known in the art.

Inhibition of the expression of an IGFALS or IGF-1 protein may bemanifested by a reduction in the level of the IGFALS or IGF-1 proteinthat is expressed by a cell or group of cells (e.g., the level ofprotein expressed in a sample derived from a subject). As explainedabove, for the assessment of mRNA suppression, the inhibition of proteinexpression levels in a treated cell or group of cells may similarly beexpressed as a percentage of the level of protein in a control cell orgroup of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of an IGFALS or IGF-1 gene includes a cellor group of cells that has not yet been contacted with an RNAi agent ofthe invention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

In certain embodiments, inhibition of expression of an IGF-1 gene may bemanifested in a reduction in the difference between a normal level ofIGF-1 mRNA or protein and an abnormal level of IGF-1 mRNA or protein ina subject or in a specific tissue in the subject, e.g., mRNA in theliver of the subject or IGF-1 protein in subject serum. That is,inhibition may be manifested in a normalization of expression ascompared to an appropriate control.

The level of IGFALS mRNA or IGF-1 mRNA that is expressed by a cell orgroup of cells, or the level of circulating IGFALS mRNA or IGF-1 mRNA,may be determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of IGFALS orIGF-1 in a sample is determined by detecting a transcribedpolynucleotide, or portion thereof, e.g., mRNA of the IGFALS gene orIGF-1 gene. RNA may be extracted from cells using RNA extractiontechniques including, for example, using acid phenol/guanidineisothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNApreparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland).Typical assay formats utilizing ribonucleic acid hybridization includenuclear run-on assays, RT-PCR, RNase protection assays, northernblotting, in situ hybridization, and microarray analysis. CirculatingIGFALS or IGF-1 mRNA may be detected using methods the described in PCTPublication WO2012/177906, the entire contents of which are herebyincorporated herein by reference.

In some embodiments, the level of expression of IGFALS or IGF-1 isdetermined using a nucleic acid probe. The term “probe”, as used herein,refers to any molecule that is capable of selectively binding to aspecific IGFALS or IGF-1. Probes can be synthesized by one of skill inthe art, or derived from appropriate biological preparations. Probes maybe specifically designed to be labeled. Examples of molecules that canbe utilized as probes include, but are not limited to, RNA, DNA,proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to IGFALSmRNA or IGF-1 mRNA. In one embodiment, the mRNA is immobilized on asolid surface and contacted with a probe, for example by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In an alternative embodiment, theprobe(s) are immobilized on a solid surface and the mRNA is contactedwith the probe(s), for example, in an Affymetrix gene chip array. Askilled artisan can readily adapt known mRNA detection methods for usein determining the level of IGFALS mRNA or IGF-1 mRNA.

An alternative method for determining the level of expression of IGFALSor IGF-1 in a sample involves the process of nucleic acid amplificationand/or reverse transcriptase (to prepare cDNA) of for example mRNA inthe sample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of IGFALSor IGF-1 is determined by quantitative fluorogenic RT-PCR (i.e., theTaqMan™ System).

The expression levels of IGFALS or IGF-1 mRNA may be monitored using amembrane blot (such as used in hybridization analysis such as northern,Southern, dot, and the like), or microwells, sample tubes, gels, beadsor fibers (or any solid support comprising bound nucleic acids). SeeU.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934,which are incorporated herein by reference. The determination of IGFALSor IGF-1 expression level may also comprise using nucleic acid probes insolution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of IGFALS or IGF-1 protein expression may be determined usingany method known in the art for the measurement of protein levels. Suchmethods include, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, fluid or gelprecipitin reactions, absorption spectroscopy, a colorimetric assays,spectrophotometric assays, flow cytometry, immunodiffusion (single ordouble), immunoelectrophoresis, western blotting, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention in thetreatment of an IGF system-associated disease is assessed by a decreasein IGFALS mRNA or IGF-1 mRNA level (by liver biopsy) or IGFALS or IGF-1protein level, typically determined in serum.

In some embodiments, the efficacy of the methods of the invention in thetreatment of acromegaly can be monitored by evaluating a subject fornormalization of at least one sign or symptom of acromegaly previouslydisplayed in the subject including, elevated IGF-1 level, sleep apnea,joint pain, symptomatic carpal tunnel syndrome, hypertension,biventricular cardiac hypertrophy, cardiac arrhythmia, fatigue, andweakness. These symptoms may be assessed in vitro or in vivo using anymethod known in the art. Although the nadir GH suppression ofteradministration of glucose can be considered the “gold standard” test foracromegaly (Katznelson et al., 2011, Endocrine Practice), suppressionmay not be observed after treatment with the RNAi agents provided hereindue to their proposed mechanism of action. Moreover, subjects may haveaccomplished clinically relevant beneficial outcomes with lowering ofIGF-1 without reaching normal GH levels.

It is understood that normal IGF-1 levels are dependent both on the ageand gender of the subject, with younger subjects having lower IGF-1levels than older subjects. Therefore, when comparing IGF-1 levels todetermine the lowering or normalizing of the level, an appropriatecontrol must be selected. Appropriate controls include, for example, anIGF-1 level prior to treatment (when available) or an age and gendermatched control. In certain embodiments, IGF-1 levels are monitored ortested on multiple occasions to confirm a change in IGF-1 level in asubject. In preferred embodiments, the IGF-1 level is decreasedsufficiently to provide a clinically beneficial outcome for the subject.

In some embodiments, the efficacy of the method of the invention intreatment of cancer can be monitored by evaluating a subject formaintenance or preferably reduction of tumor burden of the primary tumoror metastatic tumor(s) or the prevention of metastasis. Methods fordetection and monitoring of tumor burden are known in the art, e.g.,RECIST criteria as provided in Eisenhauer et al., 2009, New responseevaluation criteria in solid tumours: Revised RECIST guideline (version1.1). Eur. J. Cancer. 45:228-247.

In some embodiments of the methods of the invention, the iRNA isadministered to a subject such that the iRNA is delivered to a specificsite within the subject. The inhibition of expression of IGFALS or IGF-1may be assessed using measurements of the level or change in the levelof IGFALS or IGF-1 mRNA or IGFALS or IGF-1 protein in a sample derivedfrom fluid or tissue from the specific site within the subject.

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

VII. Methods of Treating or Preventing IGF System-Associated Diseases

The present invention also provides methods of using an iRNA of theinvention or a composition containing an iRNA of the invention to reduceor inhibit IGFALS or IGF-1 expression in a cell. The methods includecontacting the cell with a dsRNA of the invention and maintaining thecell for a time sufficient to obtain degradation of the mRNA transcriptof an IGFALS gene or an IGF-1 gene, thereby inhibiting expression of theIGFALS gene or an IGF-1 gene in the cell. Reduction in gene expressioncan be assessed by any methods known in the art. For example, areduction in the expression of IGFALS or IGF-1 may be determined bydetermining the mRNA expression level of IGFALS or IGF-1, e.g., in aliver sample, using methods routine to one of ordinary skill in the art,e.g., northern blotting, qRT-PCR; by determining the protein level ofIGFALS or IGF-1 using methods routine to one of ordinary skill in theart, such as western blotting, immunological techniques. A reduction inthe expression of IGFALS or IGF-1 may also be assessed indirectly bymeasuring a decrease in biological activity of IGFALS or IGF-1 ormeasuring the level of IGF-1 in a subject sample (e.g., a serum sample).

In the methods of the invention the cell may be contacted in vitro or invivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an IGFALS or IGF-1 gene, typically a liver cell.A cell suitable for use in the methods of the invention may be amammalian cell, e.g., a primate cell (such as a human cell or anon-human primate cell, e.g., a monkey cell or a chimpanzee cell), anon-primate cell (such as a cow cell, a pig cell, a camel cell, a llamacell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster,a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, alion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell(e.g., a duck cell or a goose cell), or a whale cell. In one embodiment,the cell is a human cell, e.g., a human liver cell.

IGFALS expression or IGF-1expression is inhibited in the cell by atleast 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,or 95%, or to a level below the level of detection of the assay. IGFALSexpression or IGF-1expression is inhibited in the cell such that thedifference between the level of expression in a subject with an IGFsystem-associated disease and the normal level of expression is reduceby at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95%.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the IGFALS gene or IGF-1 gene of the mammal to be treated.When the organism to be treated is a mammal such as a human, thecomposition can be administered by any means known in the art including,but not limited to oral, intraperitoneal, or parenteral routes,including intracranial (e.g., intraventricular, intraparenchymal, andintrathecal), intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), nasal, rectal, and topical (including buccal andsublingual) administration. In certain embodiments, the compositions areadministered by intravenous infusion or injection. In certainembodiments, the compositions are administered by subcutaneousinjection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof IGFALS or IGF-1, or a therapeutic or prophylactic effect. A depotinjection may also provide more consistent serum concentrations. Depotinjections may include subcutaneous injections or intramuscularinjections. In preferred embodiments, the depot injection is asubcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an IGFALS or IGF-1 gene in a mammal. Themethods include administering to the mammal a composition comprising adsRNA that targets an IGFALS or an IGF-1 gene in a cell of the mammaland maintaining the mammal for a time sufficient to obtain degradationof the mRNA transcript of the IGFALS gene or the IGF-1 gene, therebyinhibiting expression of the IGFALS gene or the IGF-1 gene in the cell.Reduction in gene expression can be assessed by any methods known it theart and by methods, e.g. qRT-PCR, described herein. Reduction in proteinproduction can be assessed by any methods known it the art and bymethods, e.g. ELISA, described herein. In one embodiment, a punctureliver biopsy sample serves as the tissue material for monitoring thereduction in the IGFALS gene or the IGF-1 gene or protein expression.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction or inhibition of IGFALS or IGF-1expression, in a therapeutically effective amount of an iRNA targetingan IGFALS gene or an IGF-1 gene or a pharmaceutical compositioncomprising an iRNA targeting an IGFALS gene or an IGF-1 gene.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of IGFALSgene or an IGF-1 gene expression are those having a disorder of elevatedgrowth hormone, e.g., acromegaly, or a disorder of elevated insulinsignaling, e.g., cancer. In another embodiment, a subject having adisorder of elevated growth hormone has one or more signs or symptomsassociated with acromegaly or elevated growth hormone including, but notlimited to, elevated IGF-1 level, somatic enlargement (soft tissue andbony overgrowth), excessive sweating, jaw overgrowth, sleep apnea,osteoarthropathy, joint pain, symptomatic carpal tunnel syndrome,hypertension, biventricular cardiac hypertrophy, cardiac arrhythmia,fatigue, weakness, diabetes mellitus, menstrual irregularities in womenand sexual dysfunction in men, headache, and visual field loss(attributable to optic chiasmal compression) and diplopia (due tocranial nerve palsy); in conjunction with an elevated growth hormonelevel. Treatment of a subject that would benefit from a reduction orinhibition of IGFALS or IGF-1 gene expression and normalization ofgrowth hormone levels includes therapeutic treatment (e.g., of a subjectis suffering from acromegaly) and prophylactic treatment (e.g., of asubject does not meet the diagnostic criteria of acromegaly or may haveelevated or fluctuating growth hormone, or IGFALS, or IGF-1 levels, or asubject may be at risk of developing acromegaly). Treatment of a subjectthat would benefit from a reduction or inhibition of IGFALS geneexpression or IGF-1 gene expression can also include treatment ofcancer.

The invention further provides methods for the use of an iRNA or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction or inhibition of IGFALS or IGF-1expression, e.g., a subject having a disorder of elevated growthhormone, in combination with other pharmaceuticals or other therapeuticmethods, e.g., with known pharmaceuticals or known therapeutic methods,such as, for example, those which are currently employed for treatingthese disorders. For example, in certain embodiments, an iRNA targetingIGFALS or IGF-1 is administered in combination with an agent useful intreating a disorder of elevated growth hormone as described elsewhereherein.

The invention provides methods for the treatment of cancer, e.g., IGF-1dependent cancer, IGF-1 receptor positive cancer, or metastatic orpotentially metastatic cancer. In certain embodiments, the iRNAs of theinvention are used in conjunction with various standards of treatment ofcancer, e.g., chemotherapeutic agents, surgery, radiation; andcombinations thereof.

The iRNA and additional therapeutic agents may be administered at thesame time or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times or by another method known in the artor described herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target IGFALS gene or IGF-1gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18,24 hours, 28, 32, or about 36 hours. In one embodiment, expression ofthe target IGFALS gene or IGF-1 gene is decreased for an extendedduration, e.g., at least about two, three, four days or more, e.g.,about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the targetIGFALS gene or IGF-1 gene. Compositions and methods for inhibiting theexpression of these genes using iRNAs can be prepared and performed asdescribed herein.

Administration of the iRNA according to the methods of the invention mayresult in a reduction of the severity, signs, symptoms, or markers ofsuch diseases or disorders in a patient with a disorder of elevatedgrowth hormone, elevated IGFALS, elevated IGF-1, or an IGF-1 responsivetumor. By “reduction” in this context is meant a statisticallysignificant decrease in such level. The reduction can be, for example,at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, or to below the level of detection of the assay used.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker, orany other measurable parameter appropriate for a given disease beingtreated or targeted for prevention. It is well within the ability of oneskilled in the art to monitor efficacy of treatment or prevention bymeasuring any one of such parameters, or any combination of parameters.For example, efficacy of treatment of a disorder of IGF signaling may beassessed, for example, by periodic monitoring of IGF-1 or IGFALS levels,e.g., serum IGF-1 or IGFALS levels. For subjects suffering fromacromegaly a decrease in one or more signs or symptoms including, butnot limited to sleep apnea, joint pain, symptomatic carpal tunnelsyndrome, hypertension, biventricular cardiac hypertrophy, cardiacarrhythmia, fatigue, and weakness can be an indication of treatment ofacromegaly. Similarly a delay or lessening of the severity of theco-morbidities associated with acromegaly such as hypertension,hypertrophy, stroke, diabetes, and sleep apnea can demonstrate efficacyof treatment.

Efficacy of treatment of cancer can be demonstrated by stabilization ora decrease in tumor burden as demonstrated by a stabilization ordecrease in tumor burden of the primary tumor, metastatic tumors, or thedelay or prevention of tumor metastasis. Diagnostic and monitoringmethods are known in the art and are also provided herein.

Comparisons of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of aniRNA targeting IGFALS or IGF-1, or pharmaceutical composition thereof,“effective against” an IGF system-associated disorder indicates thatadministration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as a improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating IGF system-associated disorders.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale. Anypositive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period oftime, on a regular basis. In certain embodiments, after an initialtreatment regimen, the treatments can be administered on a less frequentbasis. Administration of the iRNA can reduce IGFALS or IGF-1 levels,e.g., in a cell, tissue, blood, urine, or other compartment of thepatient by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of theassay method used. It is noted that a reduction in IGFALS will notlikely result in a decrease in growth hormone levels in a subject withacromegaly. Administration of the iRNA can reduce the difference in thesubject IGF-1 levels and a normal IGF-1 level, e.g., in a cell, tissue,blood, urine, or other compartment of the patient by at least 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired daily dose of iRNA to a subject. The injections may berepeated over a period of time. The administration may be repeated on aregular basis. In certain embodiments, after an initial treatmentregimen, the treatments can be administered on a less frequent basis. Arepeat-dose regimen may include administration of a therapeutic amountof iRNA on a regular basis, such as every other day or to once a year.In certain embodiments, the iRNA is administered about once per month toabout once per quarter (i.e., about once every three months).

IX. Diagnostic Criteria and Treatment for Acromegaly

Diagnostic criteria for acromegaly are set forth in the AmericanAssociation of Clinical Endocrinologists Medical Guidelines for ClinicalPractice for the Diagnosis and Treatment of Acromegaly-2011 Update(Katznelson et al., Endocr. Pract. 17 (Suppl. 4), incorporated herein byreference. Further details and citations can be found therein.

Acromegaly is a clinical syndrome that, depending on its stage ofprogression, may not manifest with clear diagnostic features. Diagnosisshould be considered in patients with 2 or more of the followingcomorbidities: new-onset diabetes, diffuse arthralgias, new-onset ordifficult-to-control hypertension, cardiac disease includingbiventricular hypertrophy and diastolic or systolic dysfunction,fatigue, headaches, carpal tunnel syndrome, sleep apnea syndrome,diaphoresis, loss of vision, colon polyps, and progressive jawmalocclusion. A serum IGF-I level, if accompanied by a large number ofresults from age- and sex-matched normal subjects, is a good tool toassess integrated GH secretion and is excellent for diagnosis,monitoring, and especially screening. A random IGF-I value (a marker ofintegrated GH secretion) should be measured for diagnosis and formonitoring after a therapeutic intervention. Serum GH assays are notstandardized and should not be used interchangably. The nadir GHsuppression after administration of glucose has been considered the“gold standard” test for acromegaly, however, a conflict existsregarding the threshold for diagnosis. The panel recommends that GHmeasurements be performed at baseline, then every 30 minutes for a totalof 120 minutes after administration of glucose. The inability tosuppress serum GH to less than 1 ng/mL after glucose administration istypically considered the diagnostic criterion for acromegaly, however,in a consensus guideline in 2000, the diagnosis of acromegaly wasexcluded if the patient had a random GH measurement less than 0.4 ng/mLand a normal IGF-I value. Although a nadir GH concentration of less than1 ng/mL after administration of glucose is the standard recommendationfor a normal response, the 2011 panel suggests consideration of a lowernadir GH cut point at 0.4 ng/mL after glucose administration because ofthe enhanced assay sensitivity and more frequent finding of modest GHhypersecretion. A diagnosis of acromegaly will be made by one of skillin the art considering the totality of the evidence for the patientunder consideration.

Once a biochemical diagnosis of acromegaly has been made, a magneticresonance imaging (MRI) scan of the pituitary gland should be performedbecause a pituitary GH-secreting adenoma is the most common cause ofacromegaly. Visual field testing should be performed if there is opticchiasmal compression noted on the MRI or if the patient has complaintsof reduced peripheral vision. Further biochemical testing should includea serum prolactin level (to evaluate for hyperprolactinemia) andassessment of anterior and posterior pituitary function (for potentialhypopituitarism).

The goals of therapy for acromegaly are to (1) control biochemicalindices of activity, (2) control tumor size and prevent local masseffects, (3) reduce signs and symptoms of disease, (4) prevent orimprove medical comorbidities, and (5) prevent early mortality. Theprimary mode of therapy is surgery, which is recommended for allpatients with microadenomas and for all patients who have macroadenomaswith associated mass effects. In patients with macroadenomas withoutmass effects, and with low likelihood of surgical cure, a role forsurgical de-bulking of macroadenomas to improve the response tosubsequent medical therapy has been advocated, as well as primarymedical therapy alone. Medical therapy is generally used in the adjuvantsetting. Irradiation, either conventional fractionated RT orstereotactic radiosurgery, is largely relegated to an adjuvant role.Availability of specific therapeutic options and cost of theseinterventions are taken into account with decisions regarding therapy.

The goal of surgical interventions is to decrease tumor volume, therebydecreasing production of excess growth hormone and decompress the masseffect of macroadenomas on any normal remaining pituitary gland tissues,optic nerve, or surrounding critical structures. Surgical interventionscan be curative for many subjects. Surgically resected tissue should beanalyzed to understand the tumor biology to potentially provide guidancefor treatment. Biochemical analyses are also performed post-operativelyto assess the surgical outcome.

Medical therapy is used in conjunction with surgery. Studies haveprovided conflicting results regarding the benefits of treatment withmedical interventions prior to surgery to change the nature of thetumor. The iRNAs provided herein can be used at any time in conjunctionwith surgical intervention (i.e., before or after surgery).

Adjunctive medical therapy is used in patients who cannot achieve acomplete cure by surgical intervention. Medical therapies fall intothree categories: dopamine agonists, somatostatin analogs (SSAs), and aGH receptor antagonist. Each of the medical interventions presentsdifferent risks and benefits, including substantial costs of some of thetherapies.

Dopamine agonists include cabergoline and bromocriptine. The agents area good first line therapy, especially in patients with mild biochemicalactivity, as they are realtively inexpensive and orally administered.However, side effects include gastrointestinal upset, orthostatichypotension, headache, and nasal congestion.

Somatostatin analogs (SSAs) include octreotide (Sandostatin®) LAR(long-acting release, administered as an intramuscular injection) andlanreotide (Somatuline®) Autogel (administered as a deep subcutaneousdepot injection). SSAs are less convenient for use than dopamineagonists as they must be administered by injection (50 mcg three timesdaily Sandostatin® Injection subcutaneously for 2 weeks followed bySandostatin® LAR 20 mg intragluteally every 4 weeks for 3 months; or 60,90, or 120 mg of Somatuline® every 28 days by deep subcutaneousinjection). SSAs are effective in normalizing IGF-I and GH levels inapproximately 55% of patients. The clinical and biochemical responses toSSAs are inversely related to tumor size and degree of GHhypersecretion. Octreotide LAR and lanreotide Autogel have similarefficacy profiles. In patients with an inadequate response to SSAs, theaddition of cabergoline or pegvisomant (Somavert®) may be effective forfurther lowering one or both of GH and IGF-1 levels. Potential sideeffects of SSAs, include gastrointestinal upset, malabsorption,constipation, gallbladder disease, hair loss, and bradycardia.

Pegvisomant, a GH receptor antagonist, is administered by dailysubcutaneous injection. Side effects of pegvisomant, include flulikeillness, allergic reactions, and increase in liver enzymes. Patientstreated with pegvisomant must undergo routine liver enzyme tests.Because endogenous GH levels increase with pegvisomant administrationand pegvisomant may be cross-measured in GH assays, serum GH levels arenot specific and should not be monitored in patients receivingpegvisomant. Instead, serum IGF-1 levels are monitored.

Combinations of various medical therapies may be useful in the treatmentof some acromegaly patients.

Radiation therapy is used as an adjunctive treatment is patients who donot respond sufficiently to surgical or medical interventions.

Similar treatment strategies are used in children with gigantism, a typeof acromegaly, which refers to excess GH secretion that occurs duringchildhood when the growth plates are open, leading to acceleratedvertical growth.

Some of the comorbidities of acromegaly resolve upon decreasing thelevel of GH or decreasing the responsiveness of the subject to GH.However, others are not. Unlike soft tissue changes, bone enlargement isnot reversible. Surgical interventions (e.g., carpal tunnel release,joint replacement surgery), physical therapy, and analgesic medicationscan be used to treat conditions associated with bone or soft tissueovergrowth. Respiratory disorders including sleep apnea and highersusceptibility to respiratory infections can be treated with standardinterventions and preventive strategies (e.g., influenza andpneumococcal vaccinations). Cardiovascular disease, hypertension, andstroke can be managed using standard monitoring (e.g., blood pressure,cholesterol, and lipid level monitoring) and medical treatment. Subjectsshould be monitored for the development of type 2 diabetes andneoplasia, particularly colon polyps and neoplasia. Subjects should alsobe monitored for psychological complications related to the physicalchanges and deformities that can occur with the disease. As used herein,treatment can include, but does not require, resolution of theco-morbidities of acromegaly. Treatment can include, but does notrequire, prevention or reduction of the development of one or more ofthe comorbidities associated with acromegaly. As used herein, treatmentfor acromegaly can further include, but does not require, treatment ofone or more of the comorbidities associated with acromegaly.

IX. Response Evaluation Criteria and Treatment of Cancer

Methods for detection of tumors and assessment of tumor burden are wellknown in the art. For example, the Response Evaluation Criteria in SolidTumors (RECIST) guidelines were revised in 2008 and are fully set forthin Eisenhauer et al., 2009, New response evaluation criteria in solidtumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer.45:228-247. These guidelines can be used to determine if a subject hastumor regression or no tumor progression as demonstrated by a completeresponse (CR) or partial response (PR), or stable disease (SD),respectively, as provided therein for at least a sufficient time thatthe CR, PR, or SD is detected meets the threshold of treatment oreffective treatment as provided herein. A subject with only progressivedisease (PD) after administration of an iRNA provided herein is notconsidered to have a favorable response to or be effectively treated bythe iRNA. The development of PD after a period of CR, PR, or PD isunderstood as having been effectively treated by the iRNA providedherein.

It is understood that the iRNA agents provided herein can be used inconjunction with other interventions for the treatment of cancer, e.g.,surgery, chemotherapy, or radiation.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figure and Sequence Listing, are herebyincorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

IGFALS Transcripts and siRNA Design

A set of siRNAs targeting the human IGFALS, “insulin-like growth factorbinding protein, acid labile subunit”, (human: NCBI refseqID NM_004970;NCBI GeneID: 3483), as well as toxicology-species IGFALS orthologs(cynomolgus monkey: XM_005590898; mouse: NM_008340; rat, NM_053329) weredesigned using custom R and Python scripts. The human NM_004970 REFSEQmRNA has a length of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 50through position 2160 (the coding region and 3′ UTR) was determined witha linear model derived the direct measure of mRNA knockdown from morethan 20,000 distinct siRNA designs targeting a large number ofvertebrate genes. Subsets of the IGFALS siRNAs were designed withperfect or near-perfect matches between human, cynomolgus and rhesusmonkey. A further subset was designed with perfect or near-perfectmatches to mouse and rat IGFALS orthologs. For each strand of the siRNA,a custom Python script was used in a brute force search to measure thenumber and positions of mismatches between the siRNA and all potentialalignments in the target species transcriptome. Extra weight was givento mismatches in the seed region, defined here as positions 2-9 of theantisense oligonucleotide, as well the cleavage site of the siRNA,defined here as positions 10-11 of the antisense oligonucleotide. Therelative weight of the mismatches was 2.8; 1.2:1 for seed mismatches,cleavage site, and other positions up through antisense position 19.Mismatches in the first position were ignored. A specificity score wascalculated for each strand by summing the value of each weightedmismatch. Preference was given to siRNAs whose antisense score in humanand cynomolgus monkey was >=2.0 and predicted efficacy was >=50%knockdown of the IGFALS transcript.

A detailed list of the unmodified IGFALS sense and antisense strandsequences is shown in Tables 3, 6, and 12.

A detailed list of the modified IGFALS sense and antisense strandsequences is shown in Tables 5, 8, and 14.

IGF-1 Transcripts and siRNA Design

A set of siRNAs targeting the human insulin like growth factor 1, “IGF1”(human: e.g., NCBI refseqID NM_000618; NCBI GeneID: 3479), as well astoxicology-species IGF1 orthologs (cynomolgus monkey: e.g.,XM_005572039; mouse: e.g., NM_010512; rat, e.g., NM_178866) weredesigned using custom R and Python scripts. The human NM_00618 REFSEQmRNA has a length of 7366 bases.

The rationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 265through position 7366 (the coding region and 3′ UTR) was determined witha linear model derived the direct measure of mRNA knockdown from morethan 20,000 distinct siRNA designs targeting a large number ofvertebrate genes. Subsets of the IGF1 siRNAs were designed with perfector near-perfect matches between human and cynomolgus monkey. A furthersubset was designed with perfect or near-perfect matches to human,cynomolgus monkey and mouse IGF1 orthologs. A further subset wasdesigned with perfect or near-perfect matches to mouse and rat IGF1orthologs. For each strand of the siRNA, a custom Python script was usedin a brute force search to measure the number and positions ofmismatches between the siRNA and all potential alignments in the targetspecies transcriptome. Extra weight was given to mismatches in the seedregion, defined here as positions 2-9 of the antisense oligonucleotide,as well the cleavage site of the siRNA, defined here as positions 10-11of the antisense oligonucleotide. The relative weight of the mismatcheswas 2.8; 1.2:1 for seed mismatches, cleavage site, and other positionsup through antisense position 19. Mismatches in the first position wereignored. A specificity score was calculated for each strand by summingthe value of each weighted mismatch. Preference was given to siRNAswhose antisense score in human and cynomolgus monkey was >=3.0 andpredicted efficacy was >=70% knockdown of the human IGF1 transcripts.

A detailed list of the unmodified IGF-1 sense and antisense strandsequences is shown in Tables 9, 15, and 18.

A detailed list of the modified IGF-1 sense and antisense strandsequences is shown in Tables 11, 17, and 20.

siRNA Synthesis

siRNA sequences were synthesized at 1 μmol scale on a Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500 A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids),5′phosphate and other modifications are introduced using thecorresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated singlestrands was performed on a GalNAc modified CPG support. Custom CPGuniversal solid support was used for the synthesis of antisense singlestrands. Coupling time for all phosphoramidites (100 mM in acetonitrile)is 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M inacetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes(Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v).Oxidation time was 3 minutes. All sequences were synthesized with finalremoval of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagents at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with a tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection is performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 uL ofdimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent were added and thesolution was incubated for additional 20 min at 60° C. At the end ofcleavage and deprotection step, the synthesis plate was allowed to cometo room temperature and is precipitated by addition of 1 mL ofacetontile:ethanol mixture (9:1). The plates are cooled at −80 C for 2hours, superanatant was decanted carefully with the aid of a multichannel pipette. The oligonucleotide pellet was re-suspended in 20 mMNaOAc buffer and is desalted using a 5 mL HiTrap size exclusion column(GE Healthcare) on an AKTA Purifier System equipped with an A905autosampler and a Frac 950 fraction collector. Desalted samples arecollected in 96-well plates. Samples from each sequence were analyzed byLC-MS to confirm the identity, UV (260 nm) for quantification and aselected set of samples by IEX chromatography to determine purity.

Annealing of single strands was performed on a Tecan liquid handlingrobot. Equimolar mixture of sense and antisense single strands werecombined and annealed in 96 well plates. After combining thecomplementary single strands, the 96-well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 1004 in 1×PBS.

Example 2-In Vitro Screening

Cell Culture and Transfections

Hep3B (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μlof Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. Fifty μ1 of DMEMcontaining ˜5×10³ cells were then added to the siRNA mixture. Cells wereincubated for 24 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM and in some cases 1 nMfinal duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl ofLysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μ1 of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 hours 37° C. Plates were thenincubated at 81° C. for 8 minutes.

Real Time PCR

Two μ1 of cDNA were added to a master mix containing 0.5 μl of GAPDHTaqMan Probe (Hs99999905 m1), 0.5 μl IGFALS probe (HS00744047 S1) and 5μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well ina 384 well plates (Roche cat #04887301001). Real time PCR was done in aLightCycler480 Real Time PCR system (Roche). Each duplex was tested atleast two times and data were normalized to naïve cells or cellstransfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol-Hyp-(GalNAcalkyl)3 dT 2′-deoxythymidine-3′-phosphate dC2′-deoxycytidine-3′-phosphate Y44 inverted abasic DNA(2-hydroxymethyl-tetrahydrofurane- 5-phosphate) (Tgn) Thymidine-glycolnucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphate (Aam)2′-O-(N-methylacetamide)adenosine-3′-phosphate

TABLE 3 Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAsPosition SEQ Position SEQ Duplex Sense in NM_ ID Antisense in NM_ IDname name Sense Sequence 004970.2 NO name Antisense Sequence 004970.2 NOAD-62728 A-125826 ACAGAUGAGCUCAGCGUCUUU  196-216 28 A-125827AAAGACGCUGAGCUCAUCUGUGU  194-216  85 AD-62741 A-125832AGCUCAGCGUCUUUUGCAGUU  203-223 29 A-125833 AACUGCAAAAGACGCUGAGCUCA 201-223  86 AD-68729 A-138233 CUGUGGCUGGACGGCAACAAA  318-337 C21A 30A-138234 UUUGUUGCCGUCCAGCCACAGGG  316-337 C21A  87 AD-68720 A-138215GGACGGCAACAACCUCUCGUA  326-345 C21A 31 A-138216 UACGAGAGGUUGUUGCCGUCCAG 324-345 C21A  88 AD-68717 A-138209 AACCGUCUGAGCAGGCUGGAA  549-568 G21A32 A-138210 UUCCAGCCUGCUCAGACGGUUGU  547-568 G21A  89 AD-62737 A-125830GGACCUCAACCUGGGUUGGAA  558-578 33 A-125831 UUCCAACCCAGGUUGAGGUCCCA 556-578  90 AD-62713 A-125820 UGGCAACAAACUGACUUACCU  645-665 34A-125821 AGGUAAGUCAGUUUGUUGCCAGC  643-665  91 AD-62742 A-125786GGUGCUGGCGGGCAACAGGCU  678-698 35 A-125787 AGCCUGUUGCCCGCCAGCACCAG 676-698  92 AD-62719 A-125838 GCUGGACCUGAGCAGGAACGA  747-767 C21A 36A-125839 UCGUUCCUGCUCAGGUCCAGCUC  745-767 C21A  93 AD-62724 A-125840CUGGACCUGAGCAGGAACGCA  748-768 G21A 37 A-125841 UGCGUUCCUGCUCAGGUCCAGCU 746-768 G21A  94 AD-68728 A-138231 GGCCAUCAAGGCAAACGUGUU  776-795 38A-138232 AACACGUUUGCCUUGAUGGCCCG  774-795  95 AD-62717 A-125806GCAACCUCAUCGCUGCCGUGA  833-853 G21A 39 A-125807 UCACGGCAGCGAUGAGGUUGCGG 831-853 G21A  96 AD-62731 A-125796 GCUGCCGUGGCCCCGGGCGCA  844-864 C21A40 A-125797 UGCGCCCGGGGCCACGGCAGCGA  842-864 C21A  97 AD-62726 A-125794UGGCCCCGGGCGCCUUCCUGA  851-871 G21A 41 A-125795 UCAGGAAGGCGCCCGGGGCCACG 849-871 G21A  98 AD-68727 A-138229 GGGCCUGAAGGCGCUGCGAUA  872-891 G21A42 A-138230 UAUCGCAGCGCCUUCAGGCCCAG  870-891 G21A  99 AD-62715 A-125774GCGUGGCUGGCCUCCUGGAGA  911-931 G21A 43 A-125775 UCUCCAGGAGGCCAGCCACGCGG 909-931 G21A 100 AD-68710 A-138195 GGCCUCCUGGAGGACACGUUA  921-940 C21A44 A-138196 UAACGUGUCCUCCAGGAGGCCAG  919-940 C21A 101 AD-62743 A-125802UGGGCCACAACCGCAUCCGGA 1043-1063 C21A 45 A-125803 UCCGGAUGCGGUUGUGGCCCAGC1041-1063 C21A 102 AD-62711 A-125788 CCACAACCGCAUCCGGCAGCU 1047-1067 46A-125789 AGCUGCCGGAUGCGGUUGUGGCC 1045-1067 103 AD-68709 A-138193AGCUGGCUGAGCGCAGCUUUA 1066-1085 G21A 47 A-138194 UAAAGCUGCGCUCAGCCAGCUGC1064-1085 G21A 104 AD-68724 A-138223 CUCACGCUAGACCACAACCAA1110-1129 G21A 48 A-138224 UUGGUUGUGGUCUAGCGUGAGCA 1108-1129 G21A 105AD-62734 A-125782 CCUCACCAACGUGGCGGUCAU 1161-1181 49 A-125783AUGACCGCCACGUUGGUGAGGCC 1159-1181 106 AD-68111 A-135415CCUCACCAACGUGGCGGUCAU 1161-1181 50 A-135416 AUGACCGCCACGUUGGUGAGGCC1159-1181 107 AD-68719 A-138213 CACCAACGUGGCGGUCAUGAA 1166-1185 51A-138214 UUCAUGACCGCCACGUUGGUGAG 1164-1185 108 AD-68712 A-138199ACCAACGUGGCGGUCAUGAAA 1167-1186 C21A 52 A-138200 UUUCAUGACCGCCACGUUGGUGA1165-1186 C21A 109 AD-62730 A-125780 UCAUGAACCUCUCUGGGAACU 1178-1198 53A-125781 AGUUCCCAGAGAGGUUCAUGACC 1176-1198 110 AD-68711 A-138197ACCUCUCUGGGAACUGUCUCA 1186-1205 C21A 54 A-138198 UGAGACAGUUCCCAGAGAGGUUC1184-1205 C21A 111 AD-68713 A-138201 CUCUCUGGGAACUGUCUCCGA1188-1207 G21A 55 A-138202 UCGGAGACAGUUCCCAGAGAGGU 1186-1207 G21A 112AD-62732 A-125812 GAACUGUCUCCGGAACCUUCA 1194-1214 C21A 56 A-125813UGAAGGUUCCGGAGACAGUUCCC 1192-1214 C21A 113 AD-68715 A-138205GGAACUGUCUCCGGAACCUUA 1195-1214 C21A 57 A-138206 UAAGGUUCCGGAGACAGUUCCCA1193-1214 C21A 114 AD-62738 A-125784 CUGUCUCCGGAACCUUCCGGA 1197-1217 58A-125785 UCCGGAAGGUUCCGGAGACAGUU 1195-1217 115 AD-62736 A-125814GUCUCCGGAACCUUCCGGAGA 1199-1219 C21A 59 A-125815 UCUCCGGAAGGUUCCGGAGACAG1197-1219 C21A 116 AD-62712 A-125804 CGGAACCUUCCGGAGCAGGUA1204-1224 G21A 60 A-125805 UACCUGCUCCGGAAGGUUCCGGA 1202-1224 G21A 117AD-68723 A-138221 AACCUUCCGGAGCAGGUGUUA 1209-1228 C21A 61 A-138222UAACACCUGCUCCGGAAGGUUCC 1207-1228 C21A 118 AD-62739 A-125800CUUCCGGAGCAGGUGUUCCGA 1210-1230 G21A 62 A-125801 UCGGAACACCUGCUCCGGAAGGU1208-1230 G21A 119 AD-62723 A-125824 CAUCUCCAGCAUCGAAGAACA 1302-1322 63A-125825 UGUUCUUCGAUGCUGGAGAUGCU 1300-1322 120 AD-62745 A-125834CUCCAGCAUCGAAGAACAGAA 1305-1325 G21A 64 A-125835 UUCUGUUCUUCGAUGCUGGAGAU1303-1325 G21A 121 AD-62733 A-125828 CAGCAUCGAAGAACAGAGCCU 1308-1328 65A-125829 AGGCUCUGUUCUUCGAUGCUGGA 1306-1328 122 AD-68718 A-138211UUCCUCAAGGACAACGGCCUA 1329-1348 C21A 66 A-138212 UAGGCCGUUGUCCUUGAGGAAGA1327-1348 C21A 123 AD-62744 A-125818 AAGGACAACGGCCUCGUGGGA1333-1353 C21A 67 A-125819 UCCCACGAGGCCGUUGUCCUUGA 1331-1353 C21A 124AD-68721 A-138217 GCUGCUGGAGCUCGACCUGAA 1388-1407 C21A 68 A-138218UUCAGGUCGAGCUCCAGCAGCUC 1386-1407 C21A 125 AD-62727 A-125810UGACCUCCAACCAGCUCACGA 1403-1423 C21A 69 A-125811 UCGUGAGCUGGUUGGAGGUCAGG1401-1423 C21A 126 AD-62740 A-125816 CAACCAGCUCACGCACCUGCA1410-1430 C21A 70 A-125817 UGCAGGUGCGUGAGCUGGUUGGA 1408-1430 C21A 127AD-62716 A-125790 UGGAGUACCUGCUGCUCUCCA 1460-1480 C21A 71 A-125791UGGAGAGCAGCAGGUACUCCAGC 1458-1480 C21A 128 AD-62725 A-125778UGCAGCGGGCCUUCUGGCUGA 1523-1543 G21A 72 A-125779 UCAGCCAGAAGGCCCGCUGCAGG1521-1543 G21A 129 AD-62714 A-125836 GCAGCGGGCCUUCUGGCUGGA 1524-1544 73A-125837 UCCAGCCAGAAGGCCCGCUGCAG 1522-1544 130 AD-68716 A-138207UUCUGGCUGGACGUCUCGCAA 1536-1555 C21A 74 A-138208 UUGCGAGACGUCCAGCCAGAAGG1534-1555 C21A 131 AD-62721 A-125792 GGCUGGACGUCUCGCACAACA1538-1558 C21A 75 A-125793 UGUUGUGCGAGACGUCCAGCCAG 1536-1558 C21A 132AD-62718 A-125822 UCAGGAAUAACUCCUUGCAGA 1577-1597 76 A-125823UCUGCAAGGAGUUAUUCCUGAGG 1575-1597 133 AD-68722 A-138219UCAGCCUCAGGAACAACUCAA 1615-1634 C21A 77 A-138220 UUGAGUUGUUCCUGAGGCUGAGG1613-1634 C21A 134 AD-68725 A-138225 CAGCCUCAGGAACAACUCACU 1616-1635 78A-138226 AGUGAGUUGUUCCUGAGGCUGAG 1614-1635 135 AD-68714 A-138203AGCCUCAGGAACAACUCACUA 1617-1636 G21A 79 A-138204 UAGUGAGUUGUUCCUGAGGCUGA1615-1636 G21A 136 AD-62722 A-125808 UCCAGGCCAUCUGUGAGGGGA1766-1786 G21A 80 A-125809 UCCCCUCACAGAUGGCCUGGACG 1764-1786 G21A 137AD-62735 A-125798 GGGGACAGGUCCUCAGUGUCA 1956-1976 C21A 81 A-125799UGACACUGAGGACCUGUCCCCAG 1954-1976 C21A 138 AD-68731 A-138237UGUCAUCAAUUAAAGGCAAAA 2054-2073 G21A 82 A-138238 UUUUGCCUUUAAUUGAUGACAGC2052-2073 G21A 139 AD-68730 A-138235 UCAAUUAAAGGCAAAGGCAAU 2059-2078 83A-138236 AUUGCCUUUGCCUUUAAUUGAUG 2057-2078 140 AD-68726 A-138227AAAGGCAAAGGCAAUCGAAUA 2065-2084 C21A 84 A-138228 UAUUCGAUUGCCUUUGCCUUUAA2063-2084 C21A 141

TABLE 4 IGFALS Single Dose Screen in Hep3B Cells Hep3B 10 nM 10 nM 1 nM1 nM 0.1 nM 0.1 nM DuplexID Avg SD Avg SD Avg SD AD-68729 53.9 16.0 57.53.8 97.4 7.0 AD-68720 48.4 4.1 73.2 27.9 116.3 9.3 AD-68717 22.6 5.655.4 8.3 101.8 26.9 AD-62742 126.5 20.7 ND 124.7 13.1 AD-62719 106.825.1 ND 66.5 4.7 AD-62724 87.8 8.4 ND 75.1 5.7 AD-68728 56.3 7.6 78.64.4 110.6 26.0 AD-62717 98.6 1.7 ND 102.9 56.2 AD-62731 105.7 3.4 ND51.1 18.3 AD-62726 70.7 37.7 ND 93.5 8.9 AD-68727 68.9 14.6 94.8 12.8124.7 23.3 AD-62715 118.5 41.8 ND 128.4 17.9 AD-68710 91.0 34.2 91.714.2 90.5 1.1 AD-62743 81.6 18.1 ND 123.9 20.9 AD-62711 107.0 11.3 ND92.0 11.5 AD-68709 42.4 2.2 38.1 1.4 76.1 1.4 AD-68724 53.5 16.7 40.97.9 73.8 1.2 AD-62734 43.1 29.6 ND 115.2 0.3 AD-68111 29.5 14.8 37.5 0.792.1 3.9 AD-68719 45.4 18.9 59.6 5.7 108.7 25.0 AD-68712 40.8 1.9 58.04.7 97.4 13.1 AD-62730 98.4 14.2 ND 119.5 17.2 AD-68711 97.3 23.0 86.65.7 110.7 14.8 AD-68713 79.6 10.0 93.6 23.9 103.3 4.2 AD-62732 89.0 13.3ND 66.0 14.3 AD-68715 48.7 13.1 71.9 6.8 78.7 7.2 AD-62738 133.2 34.5 ND123.4 13.0 AD-62736 113.6 22.9 ND 84.9 13.9 AD-62712 68.5 22.6 ND 96.925.6 AD-68723 83.3 14.5 84.4 13.8 71.8 25.4 AD-62739 99.4 13.8 ND 105.624.4 AD-68718 83.1 6.8 78.2 0.1 119.8 36.8 AD-62744 98.8 10.4 ND 139.124.7 AD-68721 61.8 8.0 81.3 4.2 99.2 14.2 AD-62727 91.6 25.9 ND 86.646.5 AD-62740 138.9 16.0 ND 117.3 55.5 AD-62716 81.5 0.2 ND 127.8 20.9AD-62725 109.1 6.1 ND 103.8 17.0 AD-62714 73.9 8.9 ND 64.0 16.0 AD-6871618.2 0.3 28.6 17.6 40.2 10.8 AD-62721 62.0 7.7 ND 91.7 6.1 AD-68722 19.20.5 51.1 0.7 53.4 22.2 AD-68725 20.3 7.5 23.0 6.2 67.6 15.6 AD-6871458.9 2.8 73.5 19.0 92.6 8.9 AD-62722 120.7 69.9 ND 115.0 25.4 AD-6273560.2 29.8 ND 100.2 22.6 AD-68731 33.5 27.2 11.8 0.9 26.6 7.4 AD-6873014.5 0.9 24.5 7.5 44.4 8.3 AD-68726 17.0 10.0 28.6 11.8 64.5 3.7AD-62728 46.3 20.5 ND 49.5 10.2 AD-62741 116.8 48.6 ND 143.6 35.9AD-62737 94.7 8.6 ND 75.2 55.1 AD-62713 83.0 20.3 ND 89.3 31.9 AD-62723103.5 32.2 ND 66.5 22.2 AD-62745 66.7 4.4 ND 85.7 61.2 AD-62733 107.11.3 ND 35.9 3.8 AD-62718 129.6 42.7 ND 87.7 39.2 AD-1955 102.5 25.0 Mock103.0 18.8 Naïve 118.0 23.5Data are expressed as percent message remaining relative to AD-1955non-targeting control.

TABLE 5 Modified Sequences Sense SEQ Antisense SEQ Start Duplex Oligo IDOligo ID Position in Nam Name Sense Sequence NO Name Antisense SequenceNO SEQ ID NO: 1 AD-68729 A-138233 csusguggCfuGfGfAfcggcaacaaaL96 142A-138234 usUfsuguUfgCfCfguccAfgCfcacagsgsg 199  316 AD-68720 A-138215gsgsacggCfaAfCfAfaccucucguaL96 143 A-138216usAfscgaGfaGfGfuuguUfgCfcguccsasg 200  324 AD-68717 A-138209asasccguCfuGfAfGfcaggcuggaaL96 144 A-138210usUfsccaGfcCfUfgcucAfgAfcgguusgsu 201  547 AD-62742 A-125786GfsgsUfgCfuGfgCfGfGfgCfaAfcAfgGfcUfL96 145 A-125787asGfscCfuGfuUfgCfccgCfcAfgCfaCfcsasg 202  676 AD-62719 A-125838GfscsUfgGfaCfcUfGfAfgCfaGfgAfaCfgAfL96 146 A-125839usCfsgUfuCfcUfgCfucaGfgUfcCfaGfcsusc 203  745 AD-62724 A-125840CfsusGfgAfcCfuGfAfGfcAfgGfaAfcGfcAfL96 147 A-125841usGfscGfuUfcCfuGfcucAfgGfuCfcAfgscsu 204  746 AD-68728 A-138231gsgsccauCfaAfGfGfcaaacguguuL96 148 A-138232asAfscacGfuUTUfgccuUfgAfuggccscsg 205  774 AD-62717 A-125806GfscsAfaCfcUfcAfUfCfgCfuGfcCfgUfgAfL96 149 A-125807usCfsaCfgGfcAfgCfgauGfaGfgUfuGfcsgsg 206  831 AD-62731 A-125796GfscsUfgCfcGfuGfGfCfcCfcGfgGfcGfcAfL96 150 A-125797usGfscGfcCfcGfgGfgccAfcGfgCfaGfcsgsa 207  842 AD-62726 A-125794UfsgsGfcCfcCfgGfGfCfgCfcUfuCfcUfgAfL96 151 A-125795usCfsaGfgAfaGfgCfgccCfgGfgGfcCfascsg 208  849 AD-68727 A-138229gsgsgccuGfaAfGfGfcgcugcgauaL96 152 A-138230usAfsucgCfaGfCfgccuUfcAfggcccsasg 209  870 AD-62715 A-125774GfscsGfuGfgCfuGfGfCfcUfcCfuGfgAfgAfL96 153 A-125775usCfsuCfcAfgGfaGfgccAfgCfcAfcGfcsgsg 210  909 AD-68710 A-138195gsgsccucCfuGfGfAfggacacguuaL96 154 A-138196usAfsacgUfgUfCfcuccAfgGfaggccsasg 211  919 AD-62743 A-125802UfsgsGfgCfcAfcAfAfCfcGfcAfuCfcGfgAfL96 155 A-125803usCfscGfgAfuGfcGfguuGfuGfgCfcCfasgsc 212 1041 AD-62711 A-125788CfscsAfcAfaCfcGfCfAfuCfcGfgCfaGfcUfL96 156 A-125789asGfscUfgCfcGfgAfugeGfgUfuGfuGfgscsc 213 1045 AD-68709 A-138193asgscuggCfuGfAfGfcgcagcuuuaL96 157 A-138194usAfsaagCfuGfCfgcucAfgCfcagcusgsc 214 1064 AD-68724 A-138223csuscacgCfuAfGfAfccacaaccaaL96 158 A-138224usUfsgguUfgUfGfgucuAfgCfgugagscsa 215 1108 AD-62734 A-125782CfscsUfcAfcCfaAfCfGfuGfgCfgGfuCfaUfL96 159 A-125783asUfsgAfcCfgCfcAfcguUfgGfuGfaGfgscsc 216 1159 AD-68111 A-135415cscsucacCfaAfCfGfuggeggucauL96 160 A-135416asUfsgacCfgCfCfacguUfgGfugaggscsc 217 1159 AD-68719 A-138213csasccaaCfgUfGfGfcggucaugaaL96 161 A-138214usUfscauGfaCfCfgccaCfgUfuggugsasg 218 1164 AD-68712 A-138199ascscaacGfuGfGfCfggucaugaaaL96 162 A-138200usUfsucaUfgAfCfcgccAfcGfuuggusgsa 219 1165 AD-62730 A-125780UfscsAfuGfaAfcCfUfCfuCfuGfgGfaAfcUfL96 163 A-125781asGfsuUfcCfcAfgAfgagGfuUfcAfuGfascsc 220 1176 AD-68711 A-138197ascscucuCfuGfGfGfaacugucucaL96 164 A-138198usGfsagaCfaGfUfucccAfgAfgaggususc 221 1184 AD-68713 A-138201csuscucuGfgGfAfAfcugucuccgaL96 165 A-138202usCfsggaGfaCfAfguucCfcAfgagagsgsu 222 1186 AD-62732 A-125812GfsasAfcUfgUfcUfCfCfgGfaAfcCfuUTcAfL96 166 A-125813usGfsaAfgGfuUfcCfggaGfaCfaGfuUfcscsc 223 1192 AD-68715 A-138205gsgsaacuGfuCfUfCfcggaaccuuaL96 167 A-138206usAfsaggUfuCfCfggagAfcAfguuccscsa 224 1193 AD-62738 A-125784CfsusGfuCfuCfcGfGfAfaCfcUfuCfcGfgAfL96 168 A-125785usCfscGfgAfaGfgUfuccGfgAfgAfcAfgsusu 225 1195 AD-62736 A-125814GfsusCfuCfcGfgAfAfCfcUfuCfcGfgAfgAfL96 169 A-125815usCfsuCfcGfgAfaGfguuCfcGfgAfgAfcsasg 226 1197 AD-62712 A-125804CfsgsGfaAfcCfuUfCfCfgGfaGfcAfgGfuAfL96 170 A-125805usAfscCfuGfcUfcCfggaAfgGfuUfcCfgsgsa 227 1202 AD-68723 A-138221asasccuuCfcGfGfAfgcagguguuaL96 171 A-138222usAfsacaCfcUfGfcuccGfgAfagguuscsc 228 1207 AD-62739 A-125800CfsusUfcCfgGfaGfCfAfgGfuGfuUfcCfgAfL96 172 A-125801usCfsgGfaAfcAfcCfugcUfcCfgGfaAfgsgsu 229 1208 AD-68718 A-138211ususccucAfaGfGfAfcaacggccuaL96 173 A-138212usAfsggcCfgUfUfguccUfuGfaggaasgsa 230 1327 AD-62744 A-125818AfsasGfgAfcAfaCfGfGfcCfuCfgUfgGfgAfL96 174 A-125819usCfscCfaCfgAfgGfccgUfuGfuCfcUfusgsa 231 1331 AD-68721 A-138217gscsugcuGfgAfGfCfucgaccugaaL96 175 A-138218usUfscagGfuCfGfagcuCfcAfgcagcsusc 232 1386 AD-62727 A-125810UfsgsAfcCfuCfcAfAfCfcAfgCfuCfaCfgAfL96 176 A-125811usCfsgUfgAfgCfuGfguuGfgAfgGfuCfasgsg 233 1401 AD-62740 A-125816CfsasAfcCfaGfcUfCfAfcGfcAfcCfuGfcAfL96 177 A-125817usGfscAfgGfuGfcGfugaGfcUfgGfuUfgsgsa 234 1408 AD-62716 A-125790UfsgsGfaGfuAfcCfUfGfcUfgCfuCfuCfcAfL96 178 A-125791usGfsgAfgAfgCfaGfcagGfuAfcUfcCfasgsc 235 1458 AD-62725 A-125778UfsgsCfaGfcGfgGfCfCfdacUfgGfcUfgAfL96 179 A-125779usCfsaGfcCfaGfaAfggcCfcGfcUfgCfasgsg 236 1521 AD-62714 A-125836GfscsAfgCfgGfgCfCfUfuCfuGfgCfuGfgAfL96 180 A-125837usCfscAfgCfcAfgAfaggCfcCfgCfuGfcsasg 237 1522 AD-68716 A-138207ususcuggCfuGfGfAfcgucucgcaaL96 181 A-138208usUfsgcgAfgAfCfguccAfgCfcagaasgsg 238 1534 AD-62721 A-125792GfsgsCfuGfgAfcGfUfCfuCfgCfaCfaAfcAfL96 182 A-125793usGfsuUfgUfgCfgAfgacGfuCfcAfgCfcsasg 239 1536 AD-68722 A-138219uscsagccUfcAfGfGfaacaacucaaL96 183 A-138220usUfsgagUfuGfUfuccuGfaGfgcugasgsg 240 1613 AD-68725 A-138225csasgccuCfaGfGfAfacaacucacuL96 184 A-138226asGfsugaGfuUfGfuuccUfgAfggcugsasg 241 1614 AD-68714 A-138203asgsccucAfgGfAfAfcaacucacuaL96 185 A-138204usAfsgugAfgUfUfguucCfuGfaggcusgsa 242 1615 AD-62722 A-125808UfscsCfaGfgCfcAfUfCfuGfuGfaGfgGfgAfL96 186 A-125809usCfscCfcUfcAfcAfgauGfgCfcUfgGfascsg 243 1764 AD-62735 A-125798GfsgsGfgAfcAfgGfUfCfcUfcAfgUfgUfcAfL96 187 A-125799usGfsaCfaCfuGfaGfgacCfuGfuCfcCfcsasg 244 1954 AD-68731 A-138237usgsucauCfaAfUfUfaaaggcaaaaL96 188 A-138238usUfsuugCfcUfUfuaauUfgAfugacasgsc 245 2052 AD-68730 A-138235uscsaauuAfaAfGfGfcaaaggcaauL96 189 A-138236asUfsugcCfuUfUfgccuUfuAfauugasusg 246 2057 AD-68726 A-138227asasaggcAfaAfGfGfcaaucgaauaL96 190 A-138228usAfsuucGfaUfUfgccuUfuGfccuuusasa 247 2063 AD-62728 A-125826AfscsAfgAfuGfaGfCfUfcAfgCfgUfcUTuUfL96 191 A-125827asAfsaGfaCfgCfuGfagcUfcAfuCfuGfusgsu 248  774 AD-62741 A-125832AfsgsCfuCfaGfcGfUfCfuUfuUfgCfaGfuUfL96 192 A-125833asAfscUfgCfaAfaAfgacGfcUfgAfgCfuscsa 249  201 AD-62737 A-125830GfsgsAfcCfuCfaAfCfCfuGfgGfuUfgGfaAfL96 193 A-125831usUfscCfaAfcCfcAfgguUfgAfgGfuCfcscsa 250  556 AD-62713 A-125820UfsgsGfcAfaCfaAfAfCfuGfaCfuUfaCfcUfL96 194 A-125821asGfsgUfaAfgUfcAfguuUfgUfuGfcCfasgsc 251  643 AD-62723 A-125824CfsasUfcUfcCfaGfCfAfuCfgAfaGfaAfcAfL96 195 A-125825usGfsuUfcUfuCfgAfugcUfgGfaGfaUfgscsu 252 1300 AD-62745 A-125834CfsusCfcAfgCfaUfCfGfaAfgAfaCfaGfaAfL96 196 A-125835usUfscUfgUfuCfuUfcgaUfgCfuGfgAfgsasu 253 1301 AD-62733 A-125828CfsasGfcAfuCfgAfAfGfaAfcAfgAfgCfcUfL96 197 A-125829asGfsgCfuCfuGfuUfcuuCfgAfuGfcUfgsgsa 254 1306 AD-62718 A-125822UfscsAfgGfaAfuAfAfCfuCfcUfuGfcAfgAfL96 198 A-125823usCfsuGfcAfaGfgAfguuAfuUfcCfuGfasgsg 255 1575

Example 3-Knockdown of IGFALs Expression with an IGFALS siRNA DecreasesExpression of IGF-1

A series of siRNAs targeting mouse IGFALS were designed and tested forthe ability to knockdown expression of IGFALs mRNA in 6-8 week oldC57Bl/6 female mice (n=3 per group). A single 10 mg/kg dose of AD-62713,AD-62724, AD-62745, or AD-62728; or PBS control, was administeredsubcutaneously on day 1. On day 7, the mice were sacrificed to assessknockdown of IGFLALS mRNA in liver and IGFALS and IGF-1 protein inserum.

AD-62728 was found to be most effective in decreasing expression ofIGFALS mRNA and protein. Specifically, at day 7, IFGALS mRNA expressionin the liver was found to be about 15% of the PBS control. At day 7after treatment with AD-62713 and AD-62745, IGFALS mRNA expression inthe liver was found to be about 65% of the PBS control for bothduplexes.

A decrease in serum IGFALS protein levels was found to correspond to thedecrease in IGFALS mRNA in the liver. Specifically, AD-62728 decreasedthe serum IGFALS protein level to about 3.9 μg/ml, as compared to about6.4 μg/ml in the PBS control. AD-62713 and AD-62745 decreased the serumIGFALS level to about 5.2 μg/ml and 4.6 μg/ml, respectively.

A decrease in serum IGF-1 was also observed in response to treatmentwith the duplexes. Specifically, AD-62727 decreased the serum IGF-1protein level to about 13 ng/ml as compared to about 34 ng/ml in the PBScontrol. AD-62713 and AD-62745 decreased serum IGF-1 levels to about 20ng/ml and 27 ng/ml, respectively.

Further, in a multidose study, AD-62728 was demonstrated to be effectivein knockdown of expression of IGFALS mRNA in liver in an expected doseresponse manner. Specifically, C57Bl/6 female mice, 6-8 weeks of age(n=3 per group) were administered either four doses of AD-62728 at 1mg/kg or 3 mg/kg once weekly, or two doses at 3 mg/kg or 10 mg/kg everyother week; or a PBS control. IGFALS mRNA knockdown was observed in theexpected dose response manner.

Example 4-In Vitro Screening

Bioinformatics

A set of double stranded RNAi agents targeting human IGFALS (human NCBIref seq ID: NM_004970; NCBI GeneID: 3483, SEQ ID NO: 1) were designedusing custom R and Python scripts. The human IGFALS REFSEQ mRNA has alength of 2168 bases.

The rationale and method for the set of agent designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 10through position 2168 was determined with a linear model derived thedirect measure of mRNA knockdown from more than 20,000 distinct siRNAdesigns targeting a large number of vertebrate genes. The custom Pythonscript built the set of agents by systematically selecting a siRNA every11 bases along the target mRNA starting at position 10. At each of thepositions, the neighboring agent (one position to the 5′ end of themRNA, one position to the 3′ end of the mRNA) was swapped into thedesign set if the predicted efficacy was better than the efficacy at theexact every-11th siRNA. Low complexity agents, i.e., those with ShannonEntropy measures below 1.35 were excluded from the set.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Dual-Glo®Luciferase constructs were generated in the psiCHECK2 plasmid andcontained approximately 2.0 kb (human) IGFALS sequences (SEQ ID NO: 23).Dual-luciferase plasmids were co-transfected with double stranded agentsinto 3000 cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif.cat #13778-150). For each well of a 384 well plate, 0.1 μl ofLipofectamine was added to 3 ng of plasmid vector and agent in 15 μl ofOpti-MEM and allowed to complex at room temperature for 15 minutes. Themixture was then added to the cells resuspended in 35 ul of freshcomplete media. Cells were incubated for 48 hours before luciferase wasmeasured. Single dose experiments were performed at 10 nM final duplexconcentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly(transfection control) and Renilla (fused to IGFALS target sequence in3′ UTR, SEQ ID NO: 23) luciferase were measured. First, media wasremoved from cells. Then Firefly luciferase activity was measured byadding 20 μl of Dual-Glo® Luciferase Reagent mixed with 20 μl ofcomplete media to each well. The mixture was incubated at roomtemperature for 30 minutes before luminescense (500 nm) was measured ona Spectramax (Molecular Devices) to detect the Firefly luciferasesignal. Renilla luciferase activity was measured by adding 20 ul of roomtemperature of Dual-Glo® Stop & Glo® Reagent to each well and the plateswere incubated for 20 minutes before luminescence was again measured todetermine the Renilla luciferase signal. The Dual-Glo® Stop & Glo®Reagent quenched the firefly luciferase signal and sustainedluminescence for the Renilla luciferase reaction. Double stranded RNAiagent activity was determined by normalizing the Renilla (IGFALS) signalto the Firefly (control) signal within each well. The magnitude of agentactivity was then assessed relative to cells that were transfected withthe same vector but were not treated with agent or were treated with anon-targeting double stranded RNAi agent. All transfections were done inquadruplicates.

TABLE 6 Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAsSense SEQ Antisense SEQ Duplex Oligo ID Oligo ID Range in  Name NameSense oligo sequence NO Range Name Antisense oligo sequence NOSEQ ID NO: 1 AD-73764 A-147667 AGGGCAGGGGUGGCCGGCA 256   11-29 A-147668UGCCGGCCACCCCUGCCCU 441   11-29 AD-73765 A-147669 CCGGCACAGCAGACGUACA257   24-42 A-147670 UGUACGUCUGCUGUGCCGG 442   24-42 AD-73766 A-147671AGACGUACCCUCCCUCGCU 258   34-52 A-147672 AGCGAGGGAGGGUACGUCU 443   34-52AD-73767 A-147673 UCCCUCGCUGCCUGCCUGA 259   44-62 A-147674UCAGGCAGGCAGCGAGGGA 444   44-62 AD-73768 A-147675 UGCCUGCAGCCUGCCCUGA260   56-74 A-147676 UCAGGGCAGGCUGCAGGCA 445   56-74 AD-73769 A-147677UGCCCUGCAUGCAGGAUGA 261   67-85 A-147678 UCAUCCUGCAUGCAGGGCA 446   67-85AD-73770 A-147679 AGGAUGGCCCUGAGGAAAG 262   79-97 A-147680CUUUCCUCAGGGCCAUCCU 447   79-97 AD-73771 A-147681 CUGAGGAAAGGAGGCCUGA263   88-106 A-147682 UCAGGCCUCCUUUCCUCAG 448   88-106 AD-73772 A-147683AGGCCUGGCCCUGGCGCUA 264   99-117 A-147684 UAGCGCCAGGGCCAGGCCU 449  99-117 AD-73773 A-147685 GCGCUGCUGCUGCUGUCCU 265  112-130 A-147686AGGACAGCAGCAGCAGCGC 450  112-130 AD-73774 A-147687 UGCUGUCCUGGGUGGCACU266  122-140 A-147688 AGUGCCACCCAGGACAGCA 451  122-140 AD-73775 A-147689UGGCACUGGGCCCCCGCAA 267  134-152 A-147690 UUGCGGGGGCCCAGUGCCA 452 134-152 AD-73776 A-147691 GCCCCCGCAGCCUGGAGGA 268  143-161 A-147692UCCUCCAGGCUGCGGGGGC 453  143-161 AD-73777 A-147693 UGGAGGGAGCAGACCCCGA269  155-173 A-147694 UCGGGGUCUGCUCCCUCCA 454  155-173 AD-73778 A-147695AGACCCCGGAACGCCGGGA 270  165-183 A-147696 UCCCGGCGUUCCGGGGUCU 455 165-183 AD-73779 A-147697 CGCCGGGGGAAGCCGAGGA 271  176-194 A-147698UCCUCGGCUUCCCCCGGCG 456  176-194 AD-73780 A-147699 CGAGGGCCCAGCGUGCCCA272  189-207 A-147700 UGGGCACGCUGGGCCCUCG 457  189-207 AD-73781 A-147701AGCGUGCCCGGCCGCCUGU 273  198-216 A-147702 ACAGGCGGCCGGGCACGCU 458 198-216 AD-73782 A-147703 GCCUGUGUCUGCAGCUACA 274  211-229 A-147704UGUAGCUGCAGACACAGGC 459  211-229 AD-73783 A-147705 UGCAGCUACGAUGACGACA275  220-238 A-147706 UGUCGUCAUCGUAGCUGCA 460  220-238 AD-73784 A-147707GACGACGCGGAUGAGCUCA 276  232-250 A-147708 UGAGCUCAUCCGCGUCGUC 461 232-250 AD-73785 A-147709 AUGAGCUCAGCGUCUUCUA 277  242-260 A-147710UAGAAGACGCUGAGCUCAU 462  242-260 AD-73786 A-147711 UCUUCUGCAGCUCCAGGAA278  254-272 A-147712 UUCCUGGAGCUGCAGAAGA 463  254-272 AD-73787 A-147713UCCAGGAACCUCACGCGCA 279  265-283 A-147714 UGCGCGUGAGGUUCCUGGA 464 265-283 AD-73788 A-147715 UCACGCGCCUGCCUGAUGA 280  275-293 A-147716UCAUCAGGCAGGCGCGUGA 465  275-293 AD-73789 A-147717 UGAUGGAGUCCCGGGCGGA281  288-306 A-147718 UCCGCCCGGGACUCCAUCA 466  288-306 AD-73790 A-147719CGGGCGGCACCCAAGCCCU 282  299-317 A-147720 AGGGCUUGGGUGCCGCCCG 467 299-317 AD-73791 A-147721 CAAGCCCUGUGGCUGGACA 283  310-328 A-147722UGUCCAGCCACAGGGCUUG 468  310-328 AD-73792 A-147723 UGGCUGGACGGCAACAACA284  319-337 A-147724 UGUUGUUGCCGUCCAGCCA 469  319-337 AD-73793 A-147725AACAACCUCUCGUCCGUCA 285  331-349 A-147726 UGACGGACGAGAGGUUGUU 470 331-349 AD-73794 A-147727 UCCGUCCCCCCGGCAGCCU 286  343-361 A-147728AGGCUGCCGGGGGGACGGA 471  343-361 AD-73795 A-147729 CGGCAGCCUUCCAGAACCU287  353-371 A-147730 AGGUUCUGGAAGGCUGCCG 472  353-371 AD-73796 A-147731CAGAACCUCUCCAGCCUGA 288  364-382 A-147732 UCAGGCUGGAGAGGUUCUG 473 364-382 AD-73797 A-147733 AGCCUGGGCUUCCUCAACA 289  376-394 A-147734UGUUGAGGAAGCCCAGGCU 474  376-394 AD-73798 A-147735 UUCCUCAACCUGCAGGGCA290  385-403 A-147736 UGCCCUGCAGGUUGAGGAA 475  385-403 AD-73799 A-147737CAGGGCGGCCAGCUGGGCA 291  397-415 A-147738 UGCCCAGCUGGCCGCCCUG 476 397-415 AD-73800 A-147739 AGCUGGGCAGCCUGGAGCA 292  407-425 A-147740UGCUCCAGGCUGCCCAGCU 477  407-425 AD-73801 A-147741 CUGGAGCCACAGGCGCUGA293  418-436 A-147742 UCAGCGCCUGUGGCUCCAG 478  418-436 AD-73802 A-147743CGCUGCUGGGCCUAGAGAA 294  431-449 A-147744 UUCUCUAGGCCCAGCAGCG 479 431-449 AD-73803 A-147745 CUAGAGAACCUGUGCCACA 295  442-460 A-147746UGUGGCACAGGUUCUCUAG 480  442-460 AD-73804 A-147747 UGUGCCACCUGCACCUGGA296  452-470 A-147748 UCCAGGUGCAGGUGGCACA 481  452-470 AD-73805 A-147749ACCUGGAGCGGAACCAGCU 297  464-482 A-147750 AGCUGGUUCCGCUCCAGGU 482 464-482 AD-73806 A-147751 AACCAGCUGCGCAGCCUGA 298  475-493 A-147752UCAGGCUGCGCAGCUGGUU 483  475-493 AD-73807 A-147753 CGCAGCCUGGCACUCGGCA299  484-502 A-147754 UGCCGAGUGCCAGGCUGCG 484  484-502 AD-73808 A-147755UCGGCACGUUUGCACACAA 300  497-515 A-147756 UUGUGUGCAAACGUGCCGA 485 497-515 AD-73809 A-147757 UUGCACACACGCCCGCGCU 301  506-524 A-147758AGCGCGGGCGUGUGUGCAA 486  506-524 AD-73810 A-147759 CCCGCGCUGGCCUCGCUCA302  517-535 A-147760 UGAGCGAGGCCAGCGCGGG 487  517-535 AD-73811 A-147761UCGCUCGGCCUCAGCAACA 303  529-547 A-147762 UGUUGCUGAGGCCGAGCGA 488 529-547 AD-73812 A-147763 AGCAACAACCGUCUGAGCA 304  541-559 A-147764UGCUCAGACGGUUGUUGCU 489  541-559 AD-73813 A-147767 CUGGAGGACGGGCUCUUCA305  562-580 A-147768 UGAAGAGCCCGUCCUCCAG 490  562-580 AD-73814 A-147769CUCUUCGAGGGCCUCGGCA 306  574-592 A-147770 UGCCGAGGCCCUCGAAGAG 491 574-592 AD-73815 A-147771 GGCCUCGGCAGCCUCUGGA 307  583-601 A-147772UCCAGAGGCUGCCGAGGCC 492  583-601 AD-73816 A-147773 UCUGGGACCUCAACCUCGA308  596-614 A-147774 UCGAGGUUGAGGUCCCAGA 493  596-614 AD-73817 A-147775AACCUCGGCUGGAAUAGCA 309  607-625 A-147776 UGCUAUUCCAGCCGAGGUU 494 607-625 AD-73818 A-147777 UGGAAUAGCCUGGCGGUGA 310  616-634 A-147778UCACCGCCAGGCUAUUCCA 495  616-634 AD-73819 A-147779 CGGUGCUCCCCGAUGCGGA311  629-647 A-147780 UCCGCAUCGGGGAGCACCG 496  629-647 AD-73820 A-147781GAUGCGGCGUUCCGCGGCA 312  640-658 A-147782 UGCCGCGGAACGCCGCAUC 497 640-658 AD-73821 A-147783 UUCCGCGGCCUGGGCAGCA 313  649-667 A-147784UGCUGCCCAGGCCGCGGAA 498  649-667 AD-73822 A-147785 GCAGCCUGCGCGAGCUGGU314  662-680 A-147786 ACCAGCUCGCGCAGGCUGC 499  662-680 AD-73823 A-147787GAGCUGGUGCUGGCGGGCA 315  673-691 A-147788 UGCCCGCCAGCACCAGCUC 500 673-691 AD-73824 A-147789 CUGGCGGGCAACAGGCUGA 316  682-700 A-147790UCAGCCUGUUGCCCGCCAG 501  682-700 AD-73825 A-147791 AGGCUGGCCUACCUGCAGA317  694-712 A-147792 UCUGCAGGUAGGCCAGCCU 502  694-712 AD-73826 A-147793ACCUGCAGCCCGCGCUCUU 318  704-722 A-147794 AAGAGCGCGGGCUGCAGGU 503 704-722 AD-73827 A-147795 CGCUCUUCAGCGGCCUGGA 319  716-734 A-147796UCCAGGCCGCUGAAGAGCG 504  716-734 AD-73828 A-147797 CGGCCUGGCCGAGCUCCGA320  726-744 A-147798 UCGGAGCUCGGCCAGGCCG 505  726-744 AD-73829 A-147799AGCUCCGGGAGCUGGACCU 321  737-755 A-147800 AGGUCCAGCUCCCGGAGCU 506 737-755 AD-73830 A-147801 CUGGACCUGAGCAGGAACA 322  748-766 A-147802UGUUCCUGCUCAGGUCCAG 507  748-766 AD-73831 A-147803 AGGAACGCGCUGCGGGCCA323  760-778 A-147804 UGGCCCGCAGCGCGUUCCU 508  760-778 AD-73832 A-147805CGGGCCAUCAAGGCAAACA 324  772-790 A-147806 UGUUUGCCUUGAUGGCCCG 509 772-790 AD-73833 A-147807 AAGGCAAACGUGUUCGUGA 325  781-799 A-147808UCACGAACACGUUUGCCUU 510  781-799 AD-73834 A-147809 UUCGUGCAGCUGCCCCGGA326  793-811 A-147810 UCCGGGGCAGCUGCACGAA 511  793-811 AD-73835 A-147813AGAAACUCUACCUGGACCA 327  815-833 A-147814 UGGUCCAGGUAGAGUUUCU 512 815-833 AD-73836 A-147815 CCUGGACCGCAACCUCAUA 328  825-843 A-147816UAUGAGGUUGCGGUCCAGG 513  825-843 AD-73837 A-147817 CCUCAUCGCUGCCGUGGCA329  837-855 A-147818 UGCCACGGCAGCGAUGAGG 514  837-855 AD-73838 A-147819CGUGGCCCCGGGCGCCUUA 330  849-867 A-147820 UAAGGCGCCCGGGGCCACG 515 849-867 AD-73839 A-147821 GGCGCCUUCCUGGGCCUGA 331  859-877 A-147822UCAGGCCCAGGAAGGCGCC 516  859-877 AD-73840 A-147823 UGGGCCUGAAGGCGCUGCA332  869-887 A-147824 UGCAGCGCCUUCAGGCCCA 517  869-887 AD-73841 A-147825CGCUGCGAUGGCUGGACCU 333  881-899 A-147826 AGGUCCAGCCAUCGCAGCG 518 881-899 AD-73842 A-147827 UGGACCUGUCCCACAACCA 334  893-911 A-147828UGGUUGUGGGACAGGUCCA 519  893-911 AD-73843 A-147829 CACAACCGCGUGGCUGGCA335  904-922 A-147830 UGCCAGCCACGCGGUUGUG 520  904-922 AD-73844 A-147831UGGCUGGCCUCCUGGAGGA 336  914-932 A-147832 UCCUCCAGGAGGCCAGCCA 521 914-932 AD-73845 A-147833 CCUGGAGGACACGUUCCCA 337  924-942 A-147834UGGGAACGUGUCCUCCAGG 522  924-942 AD-73846 A-147835 UUCCCCGGUCUGCUGGGCA338  937-955 A-147836 UGCCCAGCAGACCGGGGAA 523  937-955 AD-73847 A-147837UGCUGGGCCUGCGUGUGCU 339  947-965 A-147838 AGCACACGCAGGCCCAGCA 524 947-965 AD-73848 A-147839 CGUGUGCUGCGGCUGUCCA 340  958-976 A-147840UGGACAGCCGCAGCACACG 525  958-976 AD-73849 A-147841 CUGUCCCACAACGCCAUCA341  970-988 A-147842 UGAUGGCGUUGUGGGACAG 526  970-988 AD-73850 A-147843AACGCCAUCGCCAGCCUGA 342  979-997 A-147844 UCAGGCUGGCGAUGGCGUU 527 979-997 AD-73851 A-147845 AGCCUGCGGCCCCGCACCU 343  991-1009 A-147846AGGUGCGGGGCCGCAGGCU 528  991-1009 AD-73852 A-147847 CGCACCUUCAAGGACCUGA344 1003-1021 A-147848 UCAGGUCCUUGAAGGUGCG 529 1003-1021 AD-73853A-147849 AAGGACCUGCACUUCCUGA 345 1012-1030 A-147850 UCAGGAAGUGCAGGUCCUU530 1012-1030 AD-73854 A-147851 UUCCUGGAGGAGCUGCAGA 346 1024-1042A-147852 UCUGCAGCUCCUCCAGGAA 531 1024-1042 AD-73855 A-147853CUGCAGCUGGGCCACAACA 347 1036-1054 A-147854 UGUUGUGGCCCAGCUGCAG 5321036-1054 AD-73856 A-147855 CCACAACCGCAUCCGGCAA 348 1047-1065 A-147856UUGCCGGAUGCGGUUGUGG 533 1047-1065 AD-73857 A-147857 UCCGGCAGCUGGCUGAGCA349 1058-1076 A-147858 UGCUCAGCCAGCUGCCGGA 534 1058-1076 AD-73858A-147859 UGGCUGAGCGCAGCUUUGA 350 1067-1085 A-147860 UCAAAGCUGCGCUCAGCCA535 1067-1085 AD-73859 A-147861 AGCUUUGAGGGCCUGGGGA 351 1078-1096A-147862 UCCCCAGGCCCUCAAAGCU 536 1078-1096 AD-73860 A-147863UGGGGCAGCUUGAGGUGCU 352 1091-1109 A-147864 AGCACCUCAAGCUGCCCCA 5371091-1109 AD-73861 A-147865 UUGAGGUGCUCACGCUAGA 353 1100-1118 A-147866UCUAGCGUGAGCACCUCAA 538 1100-1118 AD-73862 A-147867 ACGCUAGACCACAACCAGA354 1111-1129 A-147868 UCUGGUUGUGGUCUAGCGU 539 1111-1129 AD-73863A-147869 AACCAGCUCCAGGAGGUCA 355 1123-1141 A-147870 UGACCUCCUGGAGCUGGUU540 1123-1141 AD-73864 A-147871 AGGAGGUCAAGGCGGGCGA 356 1133-1151A-147872 UCGCCCGCCUUGACCUCCU 541 1133-1151 AD-73865 A-147873CGGGCGCUUUCCUCGGCCU 357 1145-1163 A-147874 AGGCCGAGGAAAGCGCCCG 5421145-1163 AD-73866 A-147875 CUCGGCCUCACCAACGUGA 358 1156-1174 A-147876UCACGUUGGUGAGGCCGAG 543 1156-1174 AD-73867 A-147877 AACGUGGCGGUCAUGAACA359 1168-1186 A-147878 UGUUCAUGACCGCCACGUU 544 1168-1186 AD-73868A-147879 UCAUGAACCUCUCUGGGAA 360 1178-1196 A-147880 UUCCCAGAGAGGUUCAUGA545 1178-1196 AD-73869 A-147881 UCUGGGAACUGUCUCCGGA 361 1189-1207A-147882 UCCGGAGACAGUUCCCAGA 546 1189-1207 AD-73870 A-147883UCUCCGGAACCUUCCGGAA 362 1200-1218 A-147884 UUCCGGAAGGUUCCGGAGA 5471200-1218 AD-73871 A-147885 UUCCGGAGCAGGUGUUCCA 363 1211-1229 A-147886UGGAACACCUGCUCCGGAA 548 1211-1229 AD-73872 A-147887 GGUGUUCCGGGGCCUGGGA364 1221-1239 A-147888 UCCCAGGCCCCGGAACACC 549 1221-1239 AD-73873A-147889 CUGGGCAAGCUGCACAGCA 365 1234-1252 A-147890 UGCUGUGCAGCUUGCCCAG550 1234-1252 AD-73874 A-147891 UGCACAGCCUGCACCUGGA 366 1244-1262A-147892 UCCAGGUGCAGGCUGUGCA 551 1244-1262 AD-73875 A-147895CAGCUGCCUGGGACGCAUA 367 1266-1284 A-147896 UAUGCGUCCCAGGCAGCUG 5521266-1284 AD-73876 A-147897 GACGCAUCCGCCCGCACAA 368 1277-1295 A-147898UUGUGCGGGCGGAUGCGUC 553 1277-1295 AD-73877 A-147899 CGCACACCUUCACCGGCCU369 1289-1307 A-147900 AGGCCGGUGAAGGUGUGCG 554 1289-1307 AD-73878A-147901 UCACCGGCCUCUCGGGGCU 370 1298-1316 A-147902 AGCCCCGAGAGGCCGGUGA555 1298-1316 AD-73879 A-147903 UCGGGGCUCCGCCGACUCU 371 1309-1327A-147904 AGAGUCGGCGGAGCCCCGA 556 1309-1327 AD-73880 A-147905CGACUCUUCCUCAAGGACA 372 1321-1339 A-147906 UGUCCUUGAGGAAGAGUCG 5571321-1339 AD-73881 A-147907 CAAGGACAACGGCCUCGUA 373 1332-1350 A-147908UACGAGGCCGUUGUCCUUG 558 1332-1350 AD-73882 A-147909 GGCCUCGUGGGCAUUGAGA374 1342-1360 A-147910 UCUCAAUGCCCACGAGGCC 559 1342-1360 AD-73883A-147911 UUGAGGAGCAGAGCCUGUA 375 1355-1373 A-147912 UACAGGCUCUGCUCCUCAA560 1355-1373 AD-73884 A-147913 AGAGCCUGUGGGGGCUGGA 376 1364-1382A-147914 UCCAGCCCCCACAGGCUCU 561 1364-1382 AD-73885 A-147915GGGCUGGCGGAGCUGCUGA 377 1375-1393 A-147916 UCAGCAGCUCCGCCAGCCC 5621375-1393 AD-73886 A-147917 UGCUGGAGCUCGACCUGAA 378 1388-1406 A-147918UUCAGGUCGAGCUCCAGCA 563 1388-1406 AD-73887 A-147919 GACCUGACCUCCAACCAGA379 1399-1417 A-147920 UCUGGUUGGAGGUCAGGUC 564 1399-1417 AD-73888A-147921 UCCAACCAGCUCACGCACA 380 1408-1426 A-147922 UGUGCGUGAGCUGGUUGGA565 1408-1426 AD-73889 A-147923 ACGCACCUGCCCCACCGCA 381 1420-1438A-147924 UGCGGUGGGGCAGGUGCGU 566 1420-1438 AD-73890 A-147925CACCGCCUCUUCCAGGGCA 382 1432-1450 A-147926 UGCCCUGGAAGAGGCGGUG 5671432-1450 AD-73891 A-147927 UCCAGGGCCUGGGCAAGCU 383 1442-1460 A-147928AGCUUGCCCAGGCCCUGGA 568 1442-1460 AD-73892 A-147929 GCAAGCUGGAGUACCUGCU384 1454-1472 A-147930 AGCAGGUACUCCAGCUUGC 569 1454-1472 AD-73893A-147931 UACCUGCUGCUCUCCCGCA 385 1465-1483 A-147932 UGCGGGAGAGCAGCAGGUA570 1465-1483 AD-73894 A-147933 CUCUCCCGCAACCGCCUGA 386 1474-1492A-147934 UCAGGCGGUUGCGGGAGAG 571 1474-1492 AD-73895 A-147935CCGCCUGGCAGAGCUGCCA 387 1485-1503 A-147936 UGGCAGCUCUGCCAGGCGG 5721485-1503 AD-73896 A-147937 AGCUGCCGGCGGACGCCCU 388 1496-1514 A-147938AGGGCGUCCGCCGGCAGCU 573 1496-1514 AD-73897 A-147939 GACGCCCUGGGCCCCCUGA389 1507-1525 A-147940 UCAGGGGGCCCAGGGCGUC 574 1507-1525 AD-73898A-147941 CCCCUGCAGCGGGCCUUCU 390 1519-1537 A-147942 AGAAGGCCCGCUGCAGGGG575 1519-1537 AD-73899 A-147943 GGGCCUUCUGGCUGGACGU 391 1529-1547A-147944 ACGUCCAGCCAGAAGGCCC 576 1529-1547 AD-73900 A-147945UGGACGUCUCGCACAACCA 392 1541-1559 A-147946 UGGUUGUGCGAGACGUCCA 5771541-1559 AD-73901 A-147947 ACAACCGCCUGGAGGCAUU 393 1553-1571 A-147948AAUGCCUCCAGGCGGUUGU 578 1553-1571 AD-73902 A-147949 GAGGCAUUGCCCAACAGCA394 1564-1582 A-147950 UGCUGUUGGGCAAUGCCUC 579 1564-1582 AD-73903A-147951 CAACAGCCUCUUGGCACCA 395 1575-1593 A-147952 UGGUGCCAAGAGGCUGUUG580 1575-1593 AD-73904 A-147953 UUGGCACCACUGGGGCGGA 396 1585-1603A-147954 UCCGCCCCAGUGGUGCCAA 581 1585-1603 AD-73905 A-147955UGGGGCGGCUGCGCUACCU 397 1595-1613 A-147956 AGGUAGCGCAGCCGCCCCA 5821595-1613 AD-73906 A-147957 CGCUACCUCAGCCUCAGGA 398 1606-1624 A-147958UCCUGAGGCUGAGGUAGCG 583 1606-1624 AD-73907 A-147959 UCAGGAACAACUCACUGCA399 1619-1637 A-147960 UGCAGUGAGUUGUUCCUGA 584 1619-1637 AD-73908A-147961 CUCACUGCGGACCUUCACA 400 1629-1647 A-147962 UGUGAAGGUCCGCAGUGAG585 1629-1647 AD-73909 A-147963 ACCUUCACGCCGCAGCCCA 401 1639-1657A-147964 UGGGCUGCGGCGUGAAGGU 586 1639-1657 AD-73910 A-147965CAGCCCCCGGGCCUGGAGA 402 1651-1669 A-147966 UCUCCAGGCCCGGGGGCUG 5871651-1669 AD-73911 A-147967 GCCUGGAGCGCCUGUGGCU 403 1661-1679 A-147968AGCCACAGGCGCUCCAGGC 588 1661-1679 AD-73912 A-147969 CUGUGGCUGGAGGGUAACA404 1672-1690 A-147970 UGUUACCCUCCAGCCACAG 589 1672-1690 AD-73913A-147971 GGUAACCCCUGGGACUGUA 405 1684-1702 A-147972 UACAGUCCCAGGGGUUACC590 1684-1702 AD-73914 A-147973 GGGACUGUGGCUGCCCUCU 406 1694-1712A-147974 AGAGGGCAGCCACAGUCCC 591 1694-1712 AD-73915 A-147975UGCCCUCUCAAGGCGCUGA 407 1705-1723 A-147976 UCAGCGCCUUGAGAGGGCA 5921705-1723 AD-73916 A-147977 CGCUGCGGGACUUCGCCCU 408 1718-1736 A-147978AGGGCGAAGUCCCGCAGCG 593 1718-1736 AD-73917 A-147979 UUCGCCCUGCAGAACCCCA409 1729-1747 A-147980 UGGGGUUCUGCAGGGCGAA 594 1729-1747 AD-73918A-147981 CAGAACCCCAGUGCUGUGA 410 1738-1756 A-147982 UCACAGCACUGGGGUUCUG595 1738-1756 AD-73919 A-147983 UGCUGUGCCCCGCUUCGUA 411 1749-1767A-147984 UACGAAGCGGGGCACAGCA 596 1749-1767 AD-73920 A-147985CUUCGUCCAGGCCAUCUGU 412 1761-1779 A-147986 ACAGAUGGCCUGGACGAAG 5971761-1779 AD-73921 A-147987 CAUCUGUGAGGGGGACGAU 413 1773-1791 A-147988AUCGUCCCCCUCACAGAUG 598 1773-1791 AD-73922 A-147989 GGGGACGAUUGCCAGCCGA414 1783-1801 A-147990 UCGGCUGGCAAUCGUCCCC 599 1783-1801 AD-73923A-147991 CAGCCGCCCGCGUACACCU 415 1795-1813 A-147992 AGGUGUACGCGGGCGGCUG600 1795-1813 AD-73924 A-147993 CGUACACCUACAACAACAU 416 1805-1823A-147994 AUGUUGUUGUAGGUGUACG 601 1805-1823 AD-73925 A-147995AACAACAUCACCUGUGCCA 417 1816-1834 A-147996 UGGCACAGGUGAUGUUGUU 6021816-1834 AD-73926 A-147997 UGUGCCAGCCCGCCCGAGA 418 1828-1846 A-147998UCUCGGGCGGGCUGGCACA 603 1828-1846 AD-73927 A-147999 CGCCCGAGGUCGUGGGGCU419 1838-1856 A-148000 AGCCCCACGACCUCGGGCG 604 1838-1856 AD-73928A-148001 CGUGGGGCUCGACCUGCGA 420 1848-1866 A-148002 UCGCAGGUCGAGCCCCACG605 1848-1866 AD-73929 A-148003 ACCUGCGGGACCUCAGCGA 421 1859-1877A-148004 UCGCUGAGGUCCCGCAGGU 606 1859-1877 AD-73930 A-148005UCAGCGAGGCCCACUUUGA 422 1871-1889 A-148006 UCAAAGUGGGCCUCGCUGA 6071871-1889 AD-73931 A-148007 ACUUUGCUCCCUGCUGACA 423 1883-1901 A-148008UGUCAGCAGGGAGCAAAGU 608 1883-1901 AD-73932 A-148009 CCUGCUGACCAGGUCCCCA424 1892-1910 A-148010 UGGGGACCUGGUCAGCAGG 609 1892-1910 AD-73933A-148011 UCCCCGGACUCAAGCCCCA 425 1905-1923 A-148012 UGGGGCUUGAGUCCGGGGA610 1905-1923 AD-73934 A-148013 CAAGCCCCGGACUCAGGCA 426 1915-1933A-148014 UGCCUGAGUCCGGGGCUUG 611 1915-1933 AD-73935 A-148015UCAGGCCCCCACCUGGCUA 427 1927-1945 A-148016 UAGCCAGGUGGGGGCCUGA 6121927-1945 AD-73936 A-148017 ACCUGGCUCACCUUGUGCU 428 1937-1955 A-148018AGCACAAGGUGAGCCAGGU 613 1937-1955 AD-73937 A-148019 UUGUGCUGGGGACAGGUCA429 1949-1967 A-148020 UGACCUGUCCCCAGCACAA 614 1949-1967 AD-73938A-148021 GACAGGUCCUCAGUGUCCU 430 1959-1977 A-148022 AGGACACUGAGGACCUGUC615 1959-1977 AD-73939 A-148023 CAGUGUCCUCAGGGGCCUA 431 1969-1987A-148024 UAGGCCCCUGAGGACACUG 616 1969-1987 AD-73940 A-148025GGGCCUGCCCAGUGCACUU 432 1981-1999 A-148026 AAGUGCACUGGGCAGGCCC 6171981-1999 AD-73941 A-148027 UGCACUUGCUGGAAGACGA 433 1993-2011 A-148028UCGUCUUCCAGCAAGUGCA 618 1993-2011 AD-73942 A-148029 UGGAAGACGCAAGGGCCUA434 2002-2020 A-148030 UAGGCCCUUGCGUCUUCCA 619 2002-2020 AD-73943A-148031 AGGGCCUGAUGGGGUGGAA 435 2013-2031 A-148032 UUCCACCCCAUCAGGCCCU620 2013-2031 AD-73944 A-148033 GGGUGGAAGGCAUGGCGGA 436 2024-2042A-148034 UCCGCCAUGCCUUCCACCC 621 2024-2042 AD-73945 A-148035UGGCGGCCCCCCCAGCUGU 437 2036-2054 A-148036 ACAGCUGGGGGGGCCGCCA 6222036-2054 AD-73946 A-148037 CAGCUGUCAUCAAUUAAAG 438 2048-2066 A-148038CUUUAAUUGAUGACAGCUG 623 2048-2066 AD-73947 A-148039 AAUUAAAGGCAAAGGCAAU439 2059-2077 A-148040 AUUGCCUUUGCCUUUAAUU 624 2059-2077 AD-73948A-148041 AAGGCAAUCGAAUCUAAAA 440 2070-2088 A-148042 UUUUAGAUUCGAUUGCCUU625 2070-2088

TABLE 7 Human IGFALS Dual-Glo ® in vitro 10 nM screen Duplex NameAverage 10 nM STDEV 10 nM AD-73764 46.26 12.94 AD-73765 15.98 9.39AD-73766 27.71 1.81 AD-73767 29.96 5.64 AD-73768 53.53 15.85 AD-7376950.94 18.08 AD-73770 35.55 11.71 AD-73771 30.07 11.32 AD-73772 33.233.56 AD-73773 11.46 4.14 AD-73774 58.80 12.47 AD-73775 108.20 18.60AD-73776 51.88 20.74 AD-73777 30.64 7.39 AD-73778 81.00 19.34 AD-7377978.23 16.91 AD-73780 67.63 20.32 AD-73781 75.04 41.97 AD-73782 11.253.14 AD-73783 84.25 27.48 AD-73784 31.16 3.50 AD-73785 40.36 15.91AD-73786 26.61 4.91 AD-73787 37.73 13.41 AD-73788 41.39 9.64 AD-7378969.70 17.02 AD-73790 54.70 18.10 AD-73791 37.77 14.31 AD-73792 59.224.58 AD-73793 30.72 11.33 AD-73794 96.09 23.63 AD-73795 27.15 4.14AD-73796 44.57 8.83 AD-73797 22.69 5.07 AD-73798 52.76 11.72 AD-7379969.71 10.21 AD-73800 49.18 17.49 AD-73801 59.80 17.00 AD-73802 28.961.45 AD-73803 33.13 19.76 AD-73804 40.68 7.80 AD-73805 63.69 6.82AD-73806 66.25 14.80 AD-73807 48.62 17.85 AD-73808 25.07 4.32 AD-7380968.40 17.86 AD-73810 83.96 14.19 AD-73811 64.13 17.42 AD-73812 46.669.77 AD-73813 44.50 17.35 AD-73814 63.89 24.44 AD-73815 52.18 19.16AD-73816 46.10 24.18 AD-73817 47.24 12.69 AD-73818 26.52 4.62 AD-7381948.75 11.37 AD-73820 60.19 5.23 AD-73821 94.35 26.80 AD-73822 84.3836.20 AD-73823 40.82 16.47 AD-73824 73.14 20.30 AD-73825 28.56 4.59AD-73826 46.85 5.02 AD-73827 47.58 13.90 AD-73828 63.46 15.46 AD-7382995.35 32.53 AD-73830 58.41 9.47 AD-73831 76.16 9.56 AD-73832 66.65 24.27AD-73833 48.53 16.86 AD-73834 61.65 17.68 AD-73835 58.15 28.49 AD-7383626.15 4.79 AD-73837 43.30 9.38 AD-73838 74.76 20.65 AD-73839 78.85 7.72AD-73840 43.78 12.13 AD-73841 40.30 13.20 AD-73842 43.45 1.12 AD-7384347.08 8.45 AD-73844 110.22 43.07 AD-73845 53.10 20.78 AD-73846 100.0352.61 AD-73847 59.82 19.09 AD-73848 26.03 3.83 AD-73849 38.45 7.00AD-73850 86.08 20.23 AD-73851 61.41 7.67 AD-73852 53.33 19.36 AD-7385385.67 29.83 AD-73854 54.76 5.66 AD-73855 104.89 36.39 AD-73856 57.2413.36 AD-73857 63.18 12.14 AD-73858 20.59 3.73 AD-73859 42.26 7.68AD-73860 94.01 20.91 AD-73861 45.90 18.39 AD-73862 26.77 5.70 AD-7386339.07 19.21 AD-73864 59.26 14.59 AD-73865 41.82 10.07 AD-73866 60.9119.05 AD-73867 35.80 9.83 AD-73868 46.58 6.40 AD-73869 64.22 11.51AD-73870 80.14 7.20 AD-73871 60.16 20.80 AD-73872 56.05 24.26 AD-7387368.99 18.51 AD-73874 110.04 18.69 AD-73875 45.34 19.36 AD-73876 51.4117.32 AD-73877 48.52 10.40 AD-73878 114.98 63.70 AD-73879 60.09 8.24AD-73880 38.19 8.87 AD-73881 74.45 6.60 AD-73882 33.01 9.79 AD-7388334.58 16.31 AD-73884 53.88 4.17 AD-73885 40.86 12.23 AD-73886 48.8115.26 AD-73887 100.05 43.02 AD-73888 52.76 9.03 AD-73889 104.07 24.09AD-73890 34.25 10.25 AD-73891 59.05 17.53 AD-73892 43.11 18.36 AD-7389374.85 51.34 AD-73894 71.46 42.74 AD-73895 67.51 15.16 AD-73896 65.3819.16 AD-73897 113.90 19.73 AD-73898 30.88 11.29 AD-73899 71.21 20.59AD-73900 45.87 8.22 AD-73901 81.14 27.00 AD-73902 57.98 26.64 AD-7390360.87 50.48 AD-73904 144.84 56.92 AD-73905 80.06 7.93 AD-73906 25.226.98 AD-73907 33.52 8.04 AD-73908 88.78 21.09 AD-73909 94.23 19.36AD-73910 106.31 18.12 AD-73911 64.23 4.10 AD-73912 25.25 5.85 AD-7391342.38 3.07 AD-73914 38.34 6.64 AD-73915 61.19 28.72 AD-73916 71.86 28.39AD-73917 95.24 18.35 AD-73918 80.25 27.23 AD-73919 48.91 6.14 AD-7392039.40 11.01 AD-73921 57.14 12.93 AD-73922 45.90 21.00 AD-73923 56.0418.98 AD-73924 28.94 7.49 AD-73925 58.43 28.38 AD-73926 102.32 34.13AD-73927 100.65 27.38 AD-73928 85.51 11.58 AD-73929 51.54 4.93 AD-7393027.83 6.80 AD-73931 36.71 9.74 AD-73932 37.09 6.54 AD-73933 54.60 14.50AD-73934 188.17 65.46 AD-73935 77.02 12.48 AD-73936 71.96 24.59 AD-7393748.37 18.42 AD-73938 47.06 6.65 AD-73939 55.62 19.17 AD-73940 74.83 6.45AD-73941 41.91 18.36 AD-73942 87.02 43.38 AD-73943 47.56 6.76 AD-7394439.62 7.36 AD-73945 61.45 10.10 AD-73946 16.22 4.18 AD-73947 17.27 8.22AD-73948 33.62 7.51

TABLE 8 Modified Sense and Antisense Strand Sequences of IGFALS dsRNAsSense Antisense Duplex Oligo SEQ Oligo SEQ SEQ Name NameSense Oligo Sequence ID Name Antisense Oligo Seq ID mRNA target sequenceID AD-73764 A-147667 AGGGCAGGGGUGGCCGGCAdTdT 626 A-147668UGCCGGCCACCCCUGCCCUdTdT 811 AGGGCAGGGGUGGCCGGCA  996 AD-73765 A-147669CCGGCACAGCAGACGUACAdTdT 627 A-147670 UGUACGUCUGCUGUGCCGGdTdT 812CCGGCACAGCAGACGUACC  997 AD-73766 A-147671 AGACGUACCCUCCCUCGCUdTdT 628A-147672 AGCGAGGGAGGGUACGUCUdTdT 813 AGACGUACCCUCCCUCGCU  998 AD-73767A-147673 UCCCUCGCUGCCUGCCUGAdTdT 629 A-147674 UCAGGCAGGCAGCGAGGGAdTdT814 UCCCUCGCUGCCUGCCUGC  999 AD-73768 A-147675 UGCCUGCAGCCUGCCCUGAdTdT630 A-147676 UCAGGGCAGGCUGCAGGCAdTdT 815 UGCCUGCAGCCUGCCCUGC 1000AD-73769 A-147677 UGCCCUGCAUGCAGGAUGAdTdT 631 A-147678UCAUCCUGCAUGCAGGGCAdTdT 816 UGCCCUGCAUGCAGGAUGG 1001 AD-73770 A-147679AGGAUGGCCCUGAGGAAAGdTdT 632 A-147680 CUUUCCUCAGGGCCAUCCUdTdT 817AGGAUGGCCCUGAGGAAAG 1002 AD-73771 A-147681 CUGAGGAAAGGAGGCCUGAdTdT 633A-147682 UCAGGCCUCCUUUCCUCAGdTdT 818 CUGAGGAAAGGAGGCCUGG 1003 AD-73772A-147683 AGGCCUGGCCCUGGCGCUAdTdT 634 A-147684 UAGCGCCAGGGCCAGGCCUdTdT819 AGGCCUGGCCCUGGCGCUG 1004 AD-73773 A-147685 GCGCUGCUGCUGCUGUCCUdTdT635 A-147686 AGGACAGCAGCAGCAGCGCdTdT 820 GCGCUGCUGCUGCUGUCCU 1005AD-73774 A-147687 UGCUGUCCUGGGUGGCACUdTdT 636 A-147688AGUGCCACCCAGGACAGCAdTdT 821 UGCUGUCCUGGGUGGCACU 1006 AD-73775 A-147689UGGCACUGGGCCCCCGCAAdTdT 637 A-147690 UUGCGGGGGCCCAGUGCCAdTdT 822UGGCACUGGGCCCCCGCAG 1007 AD-73776 A-147691 GCCCCCGCAGCCUGGAGGAdTdT 638A-147692 UCCUCCAGGCUGCGGGGGCdTdT 823 GCCCCCGCAGCCUGGAGGG 1008 AD-73777A-147693 UGGAGGGAGCAGACCCCGAdTdT 639 A-147694 UCGGGGUCUGCUCCCUCCAdTdT824 UGGAGGGAGCAGACCCCGG 1009 AD-73778 A-147695 AGACCCCGGAACGCCGGGAdTdT640 A-147696 UCCCGGCGUUCCGGGGUCUdTdT 825 AGACCCCGGAACGCCGGGG 1010AD-73779 A-147697 CGCCGGGGGAAGCCGAGGAdTdT 641 A-147698UCCUCGGCUUCCCCCGGCGdTdT 826 CGCCGGGGGAAGCCGAGGG 1011 AD-73780 A-147699CGAGGGCCCAGCGUGCCCAdTdT 642 A-147700 UGGGCACGCUGGGCCCUCGdTdT 827CGAGGGCCCAGCGUGCCCG 1012 AD-73781 A-147701 AGCGUGCCCGGCCGCCUGUdTdT 643A-147702 ACAGGCGGCCGGGCACGCUdTdT 828 AGCGUGCCCGGCCGCCUGU 1013 AD-73782A-147703 GCCUGUGUCUGCAGCUACAdTdT 644 A-147704 UGUAGCUGCAGACACAGGCdTdT829 GCCUGUGUCUGCAGCUACG 1014 AD-73783 A-147705 UGCAGCUACGAUGACGACAdTdT645 A-147706 UGUCGUCAUCGUAGCUGCAdTdT 830 UGCAGCUACGAUGACGACG 1015AD-73784 A-147707 GACGACGCGGAUGAGCUCAdTdT 646 A-147708UGAGCUCAUCCGCGUCGUCdTdT 831 GACGACGCGGAUGAGCUCA 1016 AD-73785 A-147709AUGAGCUCAGCGUCUUCUAdTdT 647 A-147710 UAGAAGACGCUGAGCUCAUdTdT 832AUGAGCUCAGCGUCUUCUG 1017 AD-73786 A-147711 UCUUCUGCAGCUCCAGGAAdTdT 648A-147712 UUCCUGGAGCUGCAGAAGAdTdT 833 UCUUCUGCAGCUCCAGGAA 1018 AD-73787A-147713 UCCAGGAACCUCACGCGCAdTdT 649 A-147714 UGCGCGUGAGGUUCCUGGAdTdT834 UCCAGGAACCUCACGCGCC 1019 AD-73788 A-147715 UCACGCGCCUGCCUGAUGAdTdT650 A-147716 UCAUCAGGCAGGCGCGUGAdTdT 835 UCACGCGCCUGCCUGAUGG 1020AD-73789 A-147717 UGAUGGAGUCCCGGGCGGAdTdT 651 A-147718UCCGCCCGGGACUCCAUCAdTdT 836 UGAUGGAGUCCCGGGCGGC 1021 AD-73790 A-147719CGGGCGGCACCCAAGCCCUdTdT 652 A-147720 AGGGCUUGGGUGCCGCCCGdTdT 837CGGGCGGCACCCAAGCCCU 1022 AD-73791 A-147721 CAAGCCCUGUGGCUGGACAdTdT 653A-147722 UGUCCAGCCACAGGGCUUGdTdT 838 CAAGCCCUGUGGCUGGACG 1023 AD-73792A-147723 UGGCUGGACGGCAACAACAdTdT 654 A-147724 UGUUGUUGCCGUCCAGCCAdTdT839 UGGCUGGACGGCAACAACC 1024 AD-73793 A-147725 AACAACCUCUCGUCCGUCAdTdT655 A-147726 UGACGGACGAGAGGUUGUUdTdT 840 AACAACCUCUCGUCCGUCC 1025AD-73794 A-147727 UCCGUCCCCCCGGCAGCCUdTdT 656 A-147728AGGCUGCCGGGGGGACGGAdTdT 841 UCCGUCCCCCCGGCAGCCU 1026 AD-73795 A-147729CGGCAGCCUUCCAGAACCUdTdT 657 A-147730 AGGUUCUGGAAGGCUGCCGdTdT 842CGGCAGCCUUCCAGAACCU 1027 AD-73796 A-147731 CAGAACCUCUCCAGCCUGAdTdT 658A-147732 UCAGGCUGGAGAGGUUCUGdTdT 843 CAGAACCUCUCCAGCCUGG 1028 AD-73797A-147733 AGCCUGGGCUUCCUCAACAdTdT 659 A-147734 UGUUGAGGAAGCCCAGGCUdTdT844 AGCCUGGGCUUCCUCAACC 1029 AD-73798 A-147735 UUCCUCAACCUGCAGGGCAdTdT660 A-147736 UGCCCUGCAGGUUGAGGAAdTdT 845 UUCCUCAACCUGCAGGGCG 1030AD-73799 A-147737 CAGGGCGGCCAGCUGGGCAdTdT 661 A-147738UGCCCAGCUGGCCGCCCUGdTdT 846 CAGGGCGGCCAGCUGGGCA 1031 AD-73800 A-147739AGCUGGGCAGCCUGGAGCAdTdT 662 A-147740 UGCUCCAGGCUGCCCAGCUdTdT 847AGCUGGGCAGCCUGGAGCC 1032 AD-73801 A-147741 CUGGAGCCACAGGCGCUGAdTdT 663A-147742 UCAGCGCCUGUGGCUCCAGdTdT 848 CUGGAGCCACAGGCGCUGC 1033 AD-73802A-147743 CGCUGCUGGGCCUAGAGAAdTdT 664 A-147744 UUCUCUAGGCCCAGCAGCGdTdT849 CGCUGCUGGGCCUAGAGAA 1034 AD-73803 A-147745 CUAGAGAACCUGUGCCACAdTdT665 A-147746 UGUGGCACAGGUUCUCUAGdTdT 850 CUAGAGAACCUGUGCCACC 1035AD-73804 A-147747 UGUGCCACCUGCACCUGGAdTdT 666 A-147748UCCAGGUGCAGGUGGCACAdTdT 851 UGUGCCACCUGCACCUGGA 1036 AD-73805 A-147749ACCUGGAGCGGAACCAGCUdTdT 667 A-147750 AGCUGGUUCCGCUCCAGGUdTdT 852ACCUGGAGCGGAACCAGCU 1037 AD-73806 A-147751 AACCAGCUGCGCAGCCUGAdTdT 668A-147752 UCAGGCUGCGCAGCUGGUUdTdT 853 AACCAGCUGCGCAGCCUGG 1038 AD-73807A-147753 CGCAGCCUGGCACUCGGCAdTdT 669 A-147754 UGCCGAGUGCCAGGCUGCGdTdT854 CGCAGCCUGGCACUCGGCA 1039 AD-73808 A-147755 UCGGCACGUUUGCACACAAdTdT670 A-147756 UUGUGUGCAAACGUGCCGAdTdT 855 UCGGCACGUUUGCACACAC 1040AD-73809 A-147757 UUGCACACACGCCCGCGCUdTdT 671 A-147758AGCGCGGGCGUGUGUGCAAdTdT 856 UUGCACACACGCCCGCGCU 1041 AD-73810 A-147759CCCGCGCUGGCCUCGCUCAdTdT 672 A-147760 UGAGCGAGGCCAGCGCGGGdTdT 857CCCGCGCUGGCCUCGCUCG 1042 AD-73811 A-147761 UCGCUCGGCCUCAGCAACAdTdT 673A-147762 UGUUGCUGAGGCCGAGCGAdTdT 858 UCGCUCGGCCUCAGCAACA 1043 AD-73812A-147763 AGCAACAACCGUCUGAGCAdTdT 674 A-147764 UGCUCAGACGGUUGUUGCUdTdT859 AGCAACAACCGUCUGAGCA 1044 AD-73813 A-147767 CUGGAGGACGGGCUCUUCAdTdT675 A-147768 UGAAGAGCCCGUCCUCCAGdTdT 860 CUGGAGGACGGGCUCUUCG 1045AD-73814 A-147769 CUCUUCGAGGGCCUCGGCAdTdT 676 A-147770UGCCGAGGCCCUCGAAGAGdTdT 861 CUCUUCGAGGGCCUCGGCA 1046 AD-73815 A-147771GGCCUCGGCAGCCUCUGGAdTdT 677 A-147772 UCCAGAGGCUGCCGAGGCCdTdT 862GGCCUCGGCAGCCUCUGGG 1047 AD-73816 A-147773 UCUGGGACCUCAACCUCGAdTdT 678A-147774 UCGAGGUUGAGGUCCCAGAdTdT 863 UCUGGGACCUCAACCUCGG 1048 AD-73817A-147775 AACCUCGGCUGGAAUAGCAdTdT 679 A-147776 UGCUAUUCCAGCCGAGGUUdTdT864 AACCUCGGCUGGAAUAGCC 1049 AD-73818 A-147777 UGGAAUAGCCUGGCGGUGAdTdT680 A-147778 UCACCGCCAGGCUAUUCCAdTdT 865 UGGAAUAGCCUGGCGGUGC 1050AD-73819 A-147779 CGGUGCUCCCCGAUGCGGAdTdT 681 A-147780UCCGCAUCGGGGAGCACCGdTdT 866 CGGUGCUCCCCGAUGCGGC 1051 AD-73820 A-147781GAUGCGGCGUUCCGCGGCAdTdT 682 A-147782 UGCCGCGGAACGCCGCAUCdTdT 867GAUGCGGCGUUCCGCGGCC 1052 AD-73821 A-147783 UUCCGCGGCCUGGGCAGCAdTdT 683A-147784 UGCUGCCCAGGCCGCGGAAdTdT 868 UUCCGCGGCCUGGGCAGCC 1053 AD-73822A-147785 GCAGCCUGCGCGAGCUGGUdTdT 684 A-147786 ACCAGCUCGCGCAGGCUGCdTdT869 GCAGCCUGCGCGAGCUGGU 1054 AD-73823 A-147787 GAGCUGGUGCUGGCGGGCAdTdT685 A-147788 UGCCCGCCAGCACCAGCUCdTdT 870 GAGCUGGUGCUGGCGGGCA 1055AD-73824 A-147789 CUGGCGGGCAACAGGCUGAdTdT 686 A-147790UCAGCCUGUUGCCCGCCAGdTdT 871 CUGGCGGGCAACAGGCUGG 1056 AD-73825 A-147791AGGCUGGCCUACCUGCAGAdTdT 687 A-147792 UCUGCAGGUAGGCCAGCCUdTdT 872AGGCUGGCCUACCUGCAGC 1057 AD-73826 A-147793 ACCUGCAGCCCGCGCUCUUdTdT 688A-147794 AAGAGCGCGGGCUGCAGGUdTdT 873 ACCUGCAGCCCGCGCUCUU 1058 AD-73827A-147795 CGCUCUUCAGCGGCCUGGAdTdT 689 A-147796 UCCAGGCCGCUGAAGAGCGdTdT874 CGCUCUUCAGCGGCCUGGC 1059 AD-73828 A-147797 CGGCCUGGCCGAGCUCCGAdTdT690 A-147798 UCGGAGCUCGGCCAGGCCGdTdT 875 CGGCCUGGCCGAGCUCCGG 1060AD-73829 A-147799 AGCUCCGGGAGCUGGACCUdTdT 691 A-147800AGGUCCAGCUCCCGGAGCUdTdT 876 AGCUCCGGGAGCUGGACCU 1061 AD-73830 A-147801CUGGACCUGAGCAGGAACAdTdT 692 A-147802 UGUUCCUGCUCAGGUCCAGdTdT 877CUGGACCUGAGCAGGAACG 1062 AD-73831 A-147803 AGGAACGCGCUGCGGGCCAdTdT 693A-147804 UGGCCCGCAGCGCGUUCCUdTdT 878 AGGAACGCGCUGCGGGCCA 1063 AD-73832A-147805 CGGGCCAUCAAGGCAAACAdTdT 694 A-147806 UGUUUGCCUUGAUGGCCCGdTdT879 CGGGCCAUCAAGGCAAACG 1064 AD-73833 A-147807 AAGGCAAACGUGUUCGUGAdTdT695 A-147808 UCACGAACACGUUUGCCUUdTdT 880 AAGGCAAACGUGUUCGUGC 1065AD-73834 A-147809 UUCGUGCAGCUGCCCCGGAdTdT 696 A-147810UCCGGGGCAGCUGCACGAAdTdT 881 UUCGUGCAGCUGCCCCGGC 1066 AD-73835 A-147813AGAAACUCUACCUGGACCAdTdT 697 A-147814 UGGUCCAGGUAGAGUUUCUdTdT 882AGAAACUCUACCUGGACCG 1067 AD-73836 A-147815 CCUGGACCGCAACCUCAUAdTdT 698A-147816 UAUGAGGUUGCGGUCCAGGdTdT 883 CCUGGACCGCAACCUCAUC 1068 AD-73837A-147817 CCUCAUCGCUGCCGUGGCAdTdT 699 A-147818 UGCCACGGCAGCGAUGAGGdTdT884 CCUCAUCGCUGCCGUGGCC 1069 AD-73838 A-147819 CGUGGCCCCGGGCGCCUUAdTdT700 A-147820 UAAGGCGCCCGGGGCCACGdTdT 885 CGUGGCCCCGGGCGCCUUC 1070AD-73839 A-147821 GGCGCCUUCCUGGGCCUGAdTdT 701 A-147822UCAGGCCCAGGAAGGCGCCdTdT 886 GGCGCCUUCCUGGGCCUGA 1071 AD-73840 A-147823UGGGCCUGAAGGCGCUGCAdTdT 702 A-147824 UGCAGCGCCUUCAGGCCCAdTdT 887UGGGCCUGAAGGCGCUGCG 1072 AD-73841 A-147825 CGCUGCGAUGGCUGGACCUdTdT 703A-147826 AGGUCCAGCCAUCGCAGCGdTdT 888 CGCUGCGAUGGCUGGACCU 1073 AD-73842A-147827 UGGACCUGUCCCACAACCAdTdT 704 A-147828 UGGUUGUGGGACAGGUCCAdTdT889 UGGACCUGUCCCACAACCG 1074 AD-73843 A-147829 CACAACCGCGUGGCUGGCAdTdT705 A-147830 UGCCAGCCACGCGGUUGUGdTdT 890 CACAACCGCGUGGCUGGCC 1075AD-73844 A-147831 UGGCUGGCCUCCUGGAGGAdTdT 706 A-147832UCCUCCAGGAGGCCAGCCAdTdT 891 UGGCUGGCCUCCUGGAGGA 1076 AD-73845 A-147833CCUGGAGGACACGUUCCCAdTdT 707 A-147834 UGGGAACGUGUCCUCCAGGdTdT 892CCUGGAGGACACGUUCCCC 1077 AD-73846 A-147835 UUCCCCGGUCUGCUGGGCAdTdT 708A-147836 UGCCCAGCAGACCGGGGAAdTdT 893 UUCCCCGGUCUGCUGGGCC 1078 AD-73847A-147837 UGCUGGGCCUGCGUGUGCUdTdT 709 A-147838 AGCACACGCAGGCCCAGCAdTdT894 UGCUGGGCCUGCGUGUGCU 1079 AD-73848 A-147839 CGUGUGCUGCGGCUGUCCAdTdT710 A-147840 UGGACAGCCGCAGCACACGdTdT 895 CGUGUGCUGCGGCUGUCCC 1080AD-73849 A-147841 CUGUCCCACAACGCCAUCAdTdT 711 A-147842UGAUGGCGUUGUGGGACAGdTdT 896 CUGUCCCACAACGCCAUCG 1081 AD-73850 A-147843AACGCCAUCGCCAGCCUGAdTdT 712 A-147844 UCAGGCUGGCGAUGGCGUUdTdT 897AACGCCAUCGCCAGCCUGC 1082 AD-73851 A-147845 AGCCUGCGGCCCCGCACCUdTdT 713A-147846 AGGUGCGGGGCCGCAGGCUdTdT 898 AGCCUGCGGCCCCGCACCU 1083 AD-73852A-147847 CGCACCUUCAAGGACCUGAdTdT 714 A-147848 UCAGGUCCUUGAAGGUGCGdTdT899 CGCACCUUCAAGGACCUGC 1084 AD-73853 A-147849 AAGGACCUGCACUUCCUGAdTdT715 A-147850 UCAGGAAGUGCAGGUCCUUdTdT 900 AAGGACCUGCACUUCCUGG 1085AD-73854 A-147851 UUCCUGGAGGAGCUGCAGAdTdT 716 A-147852UCUGCAGCUCCUCCAGGAAdTdT 901 UUCCUGGAGGAGCUGCAGC 1086 AD-73855 A-147853CUGCAGCUGGGCCACAACAdTdT 717 A-147854 UGUUGUGGCCCAGCUGCAGdTdT 902CUGCAGCUGGGCCACAACC 1087 AD-73856 A-147855 CCACAACCGCAUCCGGCAAdTdT 718A-147856 UUGCCGGAUGCGGUUGUGGdTdT 903 CCACAACCGCAUCCGGCAG 1088 AD-73857A-147857 UCCGGCAGCUGGCUGAGCAdTdT 719 A-147858 UGCUCAGCCAGCUGCCGGAdTdT904 UCCGGCAGCUGGCUGAGCG 1089 AD-73858 A-147859 UGGCUGAGCGCAGCUUUGAdTdT720 A-147860 UCAAAGCUGCGCUCAGCCAdTdT 905 UGGCUGAGCGCAGCUUUGA 1090AD-73859 A-147861 AGCUUUGAGGGCCUGGGGAdTdT 721 A-147862UCCCCAGGCCCUCAAAGCUdTdT 906 AGCUUUGAGGGCCUGGGGC 1091 AD-73860 A-147863UGGGGCAGCUUGAGGUGCUdTdT 722 A-147864 AGCACCUCAAGCUGCCCCAdTdT 907UGGGGCAGCUUGAGGUGCU 1092 AD-73861 A-147865 UUGAGGUGCUCACGCUAGAdTdT 723A-147866 UCUAGCGUGAGCACCUCAAdTdT 908 UUGAGGUGCUCACGCUAGA 1093 AD-73862A-147867 ACGCUAGACCACAACCAGAdTdT 724 A-147868 UCUGGUUGUGGUCUAGCGUdTdT909 ACGCUAGACCACAACCAGC 1094 AD-73863 A-147869 AACCAGCUCCAGGAGGUCAdTdT725 A-147870 UGACCUCCUGGAGCUGGUUdTdT 910 AACCAGCUCCAGGAGGUCA 1095AD-73864 A-147871 AGGAGGUCAAGGCGGGCGAdTdT 726 A-147872UCGCCCGCCUUGACCUCCUdTdT 911 AGGAGGUCAAGGCGGGCGC 1096 AD-73865 A-147873CGGGCGCUUUCCUCGGCCUdTdT 727 A-147874 AGGCCGAGGAAAGCGCCCGdTdT 912CGGGCGCUUUCCUCGGCCU 1097 AD-73866 A-147875 CUCGGCCUCACCAACGUGAdTdT 728A-147876 UCACGUUGGUGAGGCCGAGdTdT 913 CUCGGCCUCACCAACGUGG 1098 AD-73867A-147877 AACGUGGCGGUCAUGAACAdTdT 729 A-147878 UGUUCAUGACCGCCACGUUdTdT914 AACGUGGCGGUCAUGAACC 1099 AD-73868 A-147879 UCAUGAACCUCUCUGGGAAdTdT730 A-147880 UUCCCAGAGAGGUUCAUGAdTdT 915 UCAUGAACCUCUCUGGGAA 1100AD-73869 A-147881 UCUGGGAACUGUCUCCGGAdTdT 731 A-147882UCCGGAGACAGUUCCCAGAdTdT 916 UCUGGGAACUGUCUCCGGA 1101 AD-73870 A-147883UCUCCGGAACCUUCCGGAAdTdT 732 A-147884 UUCCGGAAGGUUCCGGAGAdTdT 917UCUCCGGAACCUUCCGGAG 1102 AD-73871 A-147885 UUCCGGAGCAGGUGUUCCAdTdT 733A-147886 UGGAACACCUGCUCCGGAAdTdT 918 UUCCGGAGCAGGUGUUCCG 1103 AD-73872A-147887 GGUGUUCCGGGGCCUGGGAdTdT 734 A-147888 UCCCAGGCCCCGGAACACCdTdT919 GGUGUUCCGGGGCCUGGGC 1104 AD-73873 A-147889 CUGGGCAAGCUGCACAGCAdTdT735 A-147890 UGCUGUGCAGCUUGCCCAGdTdT 920 CUGGGCAAGCUGCACAGCC 1105AD-73874 A-147891 UGCACAGCCUGCACCUGGAdTdT 736 A-147892UCCAGGUGCAGGCUGUGCAdTdT 921 UGCACAGCCUGCACCUGGA 1106 AD-73875 A-147895CAGCUGCCUGGGACGCAUAdTdT 737 A-147896 UAUGCGUCCCAGGCAGCUGdTdT 922CAGCUGCCUGGGACGCAUC 1107 AD-73876 A-147897 GACGCAUCCGCCCGCACAAdTdT 738A-147898 UUGUGCGGGCGGAUGCGUCdTdT 923 GACGCAUCCGCCCGCACAC 1108 AD-73877A-147899 CGCACACCUUCACCGGCCUdTdT 739 A-147900 AGGCCGGUGAAGGUGUGCGdTdT924 CGCACACCUUCACCGGCCU 1109 AD-73878 A-147901 UCACCGGCCUCUCGGGGCUdTdT740 A-147902 AGCCCCGAGAGGCCGGUGAdTdT 925 UCACCGGCCUCUCGGGGCU 1110AD-73879 A-147903 UCGGGGCUCCGCCGACUCUdTdT 741 A-147904AGAGUCGGCGGAGCCCCGAdTdT 926 UCGGGGCUCCGCCGACUCU 1111 AD-73880 A-147905CGACUCUUCCUCAAGGACAdTdT 742 A-147906 UGUCCUUGAGGAAGAGUCGdTdT 927CGACUCUUCCUCAAGGACA 1112 AD-73881 A-147907 CAAGGACAACGGCCUCGUAdTdT 743A-147908 UACGAGGCCGUUGUCCUUGdTdT 928 CAAGGACAACGGCCUCGUG 1113 AD-73882A-147909 GGCCUCGUGGGCAUUGAGAdTdT 744 A-147910 UCUCAAUGCCCACGAGGCCdTdT929 GGCCUCGUGGGCAUUGAGG 1114 AD-73883 A-147911 UUGAGGAGCAGAGCCUGUAdTdT745 A-147912 UACAGGCUCUGCUCCUCAAdTdT 930 UUGAGGAGCAGAGCCUGUG 1115AD-73884 A-147913 AGAGCCUGUGGGGGCUGGAdTdT 746 A-147914UCCAGCCCCCACAGGCUCUdTdT 931 AGAGCCUGUGGGGGCUGGC 1116 AD-73885 A-147915GGGCUGGCGGAGCUGCUGAdTdT 747 A-147916 UCAGCAGCUCCGCCAGCCCdTdT 932GGGCUGGCGGAGCUGCUGG 1117 AD-73886 A-147917 UGCUGGAGCUCGACCUGAAdTdT 748A-147918 UUCAGGUCGAGCUCCAGCAdTdT 933 UGCUGGAGCUCGACCUGAC 1118 AD-73887A-147919 GACCUGACCUCCAACCAGAdTdT 749 A-147920 UCUGGUUGGAGGUCAGGUCdTdT934 GACCUGACCUCCAACCAGC 1119 AD-73888 A-147921 UCCAACCAGCUCACGCACAdTdT750 A-147922 UGUGCGUGAGCUGGUUGGAdTdT 935 UCCAACCAGCUCACGCACC 1120AD-73889 A-147923 ACGCACCUGCCCCACCGCAdTdT 751 A-147924UGCGGUGGGGCAGGUGCGUdTdT 936 ACGCACCUGCCCCACCGCC 1121 AD-73890 A-147925CACCGCCUCUUCCAGGGCAdTdT 752 A-147926 UGCCCUGGAAGAGGCGGUGdTdT 937CACCGCCUCUUCCAGGGCC 1122 AD-73891 A-147927 UCCAGGGCCUGGGCAAGCUdTdT 753A-147928 AGCUUGCCCAGGCCCUGGAdTdT 938 UCCAGGGCCUGGGCAAGCU 1123 AD-73892A-147929 GCAAGCUGGAGUACCUGCUdTdT 754 A-147930 AGCAGGUACUCCAGCUUGCdTdT939 GCAAGCUGGAGUACCUGCU 1124 AD-73893 A-147931 UACCUGCUGCUCUCCCGCAdTdT755 A-147932 UGCGGGAGAGCAGCAGGUAdTdT 940 UACCUGCUGCUCUCCCGCA 1125AD-73894 A-147933 CUCUCCCGCAACCGCCUGAdTdT 756 A-147934UCAGGCGGUUGCGGGAGAGdTdT 941 CUCUCCCGCAACCGCCUGG 1126 AD-73895 A-147935CCGCCUGGCAGAGCUGCCAdTdT 757 A-147936 UGGCAGCUCUGCCAGGCGGdTdT 942CCGCCUGGCAGAGCUGCCG 1127 AD-73896 A-147937 AGCUGCCGGCGGACGCCCUdTdT 758A-147938 AGGGCGUCCGCCGGCAGCUdTdT 943 AGCUGCCGGCGGACGCCCU 1128 AD-73897A-147939 GACGCCCUGGGCCCCCUGAdTdT 759 A-147940 UCAGGGGGCCCAGGGCGUCdTdT944 GACGCCCUGGGCCCCCUGC 1129 AD-73898 A-147941 CCCCUGCAGCGGGCCUUCUdTdT760 A-147942 AGAAGGCCCGCUGCAGGGGdTdT 945 CCCCUGCAGCGGGCCUUCU 1130AD-73899 A-147943 GGGCCUUCUGGCUGGACGUdTdT 761 A-147944ACGUCCAGCCAGAAGGCCCdTdT 946 GGGCCUUCUGGCUGGACGU 1131 AD-73900 A-147945UGGACGUCUCGCACAACCAdTdT 762 A-147946 UGGUUGUGCGAGACGUCCAdTdT 947UGGACGUCUCGCACAACCG 1132 AD-73901 A-147947 ACAACCGCCUGGAGGCAUUdTdT 763A-147948 AAUGCCUCCAGGCGGUUGUdTdT 948 ACAACCGCCUGGAGGCAUU 1133 AD-73902A-147949 GAGGCAUUGCCCAACAGCAdTdT 764 A-147950 UGCUGUUGGGCAAUGCCUCdTdT949 GAGGCAUUGCCCAACAGCC 1134 AD-73903 A-147951 CAACAGCCUCUUGGCACCAdTdT765 A-147952 UGGUGCCAAGAGGCUGUUGdTdT 950 CAACAGCCUCUUGGCACCA 1135AD-73904 A-147953 UUGGCACCACUGGGGCGGAdTdT 766 A-147954UCCGCCCCAGUGGUGCCAAdTdT 951 UUGGCACCACUGGGGCGGC 1136 AD-73905 A-147955UGGGGCGGCUGCGCUACCUdTdT 767 A-147956 AGGUAGCGCAGCCGCCCCAdTdT 952UGGGGCGGCUGCGCUACCU 1137 AD-73906 A-147957 CGCUACCUCAGCCUCAGGAdTdT 768A-147958 UCCUGAGGCUGAGGUAGCGdTdT 953 CGCUACCUCAGCCUCAGGA 1138 AD-73907A-147959 UCAGGAACAACUCACUGCAdTdT 769 A-147960 UGCAGUGAGUUGUUCCUGAdTdT954 UCAGGAACAACUCACUGCG 1139 AD-73908 A-147961 CUCACUGCGGACCUUCACAdTdT770 A-147962 UGUGAAGGUCCGCAGUGAGdTdT 955 CUCACUGCGGACCUUCACG 1140AD-73909 A-147963 ACCUUCACGCCGCAGCCCAdTdT 771 A-147964UGGGCUGCGGCGUGAAGGUdTdT 956 ACCUUCACGCCGCAGCCCC 1141 AD-73910 A-147965CAGCCCCCGGGCCUGGAGAdTdT 772 A-147966 UCUCCAGGCCCGGGGGCUGdTdT 957CAGCCCCCGGGCCUGGAGC 1142 AD-73911 A-147967 GCCUGGAGCGCCUGUGGCUdTdT 773A-147968 AGCCACAGGCGCUCCAGGCdTdT 958 GCCUGGAGCGCCUGUGGCU 1143 AD-73912A-147969 CUGUGGCUGGAGGGUAACAdTdT 774 A-147970 UGUUACCCUCCAGCCACAGdTdT959 CUGUGGCUGGAGGGUAACC 1144 AD-73913 A-147971 GGUAACCCCUGGGACUGUAdTdT775 A-147972 UACAGUCCCAGGGGUUACCdTdT 960 GGUAACCCCUGGGACUGUG 1145AD-73914 A-147973 GGGACUGUGGCUGCCCUCUdTdT 776 A-147974AGAGGGCAGCCACAGUCCCdTdT 961 GGGACUGUGGCUGCCCUCU 1146 AD-73915 A-147975UGCCCUCUCAAGGCGCUGAdTdT 777 A-147976 UCAGCGCCUUGAGAGGGCAdTdT 962UGCCCUCUCAAGGCGCUGC 1147 AD-73916 A-147977 CGCUGCGGGACUUCGCCCUdTdT 778A-147978 AGGGCGAAGUCCCGCAGCGdTdT 963 CGCUGCGGGACUUCGCCCU 1148 AD-73917A-147979 UUCGCCCUGCAGAACCCCAdTdT 779 A-147980 UGGGGUUCUGCAGGGCGAAdTdT964 UUCGCCCUGCAGAACCCCA 1149 AD-73918 A-147981 CAGAACCCCAGUGCUGUGAdTdT780 A-147982 UCACAGCACUGGGGUUCUGdTdT 965 CAGAACCCCAGUGCUGUGC 1150AD-73919 A-147983 UGCUGUGCCCCGCUUCGUAdTdT 781 A-147984UACGAAGCGGGGCACAGCAdTdT 966 UGCUGUGCCCCGCUUCGUC 1151 AD-73920 A-147985CUUCGUCCAGGCCAUCUGUdTdT 782 A-147986 ACAGAUGGCCUGGACGAAGdTdT 967CUUCGUCCAGGCCAUCUGU 1152 AD-73921 A-147987 CAUCUGUGAGGGGGACGAUdTdT 783A-147988 AUCGUCCCCCUCACAGAUGdTdT 968 CAUCUGUGAGGGGGACGAU 1153 AD-73922A-147989 GGGGACGAUUGCCAGCCGAdTdT 784 A-147990 UCGGCUGGCAAUCGUCCCCdTdT969 GGGGACGAUUGCCAGCCGC 1154 AD-73923 A-147991 CAGCCGCCCGCGUACACCUdTdT785 A-147992 AGGUGUACGCGGGCGGCUGdTdT 970 CAGCCGCCCGCGUACACCU 1155AD-73924 A-147993 CGUACACCUACAACAACAUdTdT 786 A-147994AUGUUGUUGUAGGUGUACGdTdT 971 CGUACACCUACAACAACAU 1156 AD-73925 A-147995AACAACAUCACCUGUGCCAdTdT 787 A-147996 UGGCACAGGUGAUGUUGUUdTdT 972AACAACAUCACCUGUGCCA 1157 AD-73926 A-147997 UGUGCCAGCCCGCCCGAGAdTdT 788A-147998 UCUCGGGCGGGCUGGCACAdTdT 973 UGUGCCAGCCCGCCCGAGG 1158 AD-73927A-147999 CGCCCGAGGUCGUGGGGCUdTdT 789 A-148000 AGCCCCACGACCUCGGGCGdTdT974 CGCCCGAGGUCGUGGGGCU 1159 AD-73928 A-148001 CGUGGGGCUCGACCUGCGAdTdT790 A-148002 UCGCAGGUCGAGCCCCACGdTdT 975 CGUGGGGCUCGACCUGCGG 1160AD-73929 A-148003 ACCUGCGGGACCUCAGCGAdTdT 791 A-148004UCGCUGAGGUCCCGCAGGUdTdT 976 ACCUGCGGGACCUCAGCGA 1161 AD-73930 A-148005UCAGCGAGGCCCACUUUGAdTdT 792 A-148006 UCAAAGUGGGCCUCGCUGAdTdT 977UCAGCGAGGCCCACUUUGC 1162 AD-73931 A-148007 ACUUUGCUCCCUGCUGACAdTdT 793A-148008 UGUCAGCAGGGAGCAAAGUdTdT 978 ACUUUGCUCCCUGCUGACC 1163 AD-73932A-148009 CCUGCUGACCAGGUCCCCAdTdT 794 A-148010 UGGGGACCUGGUCAGCAGGdTdT979 CCUGCUGACCAGGUCCCCG 1164 AD-73933 A-148011 UCCCCGGACUCAAGCCCCAdTdT795 A-148012 UGGGGCUUGAGUCCGGGGAdTdT 980 UCCCCGGACUCAAGCCCCG 1165AD-73934 A-148013 CAAGCCCCGGACUCAGGCAdTdT 796 A-148014UGCCUGAGUCCGGGGCUUGdTdT 981 CAAGCCCCGGACUCAGGCC 1166 AD-73935 A-148015UCAGGCCCCCACCUGGCUAdTdT 797 A-148016 UAGCCAGGUGGGGGCCUGAdTdT 982UCAGGCCCCCACCUGGCUC 1167 AD-73936 A-148017 ACCUGGCUCACCUUGUGCUdTdT 798A-148018 AGCACAAGGUGAGCCAGGUdTdT 983 ACCUGGCUCACCUUGUGCU 1168 AD-73937A-148019 UUGUGCUGGGGACAGGUCAdTdT 799 A-148020 UGACCUGUCCCCAGCACAAdTdT984 UUGUGCUGGGGACAGGUCC 1169 AD-73938 A-148021 GACAGGUCCUCAGUGUCCUdTdT800 A-148022 AGGACACUGAGGACCUGUCdTdT 985 GACAGGUCCUCAGUGUCCU 1170AD-73939 A-148023 CAGUGUCCUCAGGGGCCUAdTdT 801 A-148024UAGGCCCCUGAGGACACUGdTdT 986 CAGUGUCCUCAGGGGCCUG 1171 AD-73940 A-148025GGGCCUGCCCAGUGCACUUdTdT 802 A-148026 AAGUGCACUGGGCAGGCCCdTdT 987GGGCCUGCCCAGUGCACUU 1172 AD-73941 A-148027 UGCACUUGCUGGAAGACGAdTdT 803A-148028 UCGUCUUCCAGCAAGUGCAdTdT 988 UGCACUUGCUGGAAGACGC 1173 AD-73942A-148029 UGGAAGACGCAAGGGCCUAdTdT 804 A-148030 UAGGCCCUUGCGUCUUCCAdTdT989 UGGAAGACGCAAGGGCCUG 1174 AD-73943 A-148031 AGGGCCUGAUGGGGUGGAAdTdT805 A-148032 UUCCACCCCAUCAGGCCCUdTdT 990 AGGGCCUGAUGGGGUGGAA 1175AD-73944 A-148033 GGGUGGAAGGCAUGGCGGAdTdT 806 A-148034UCCGCCAUGCCUUCCACCCdTdT 991 GGGUGGAAGGCAUGGCGGC 1176 AD-73945 A-148035UGGCGGCCCCCCCAGCUGUdTdT 807 A-148036 ACAGCUGGGGGGGCCGCCAdTdT 992UGGCGGCCCCCCCAGCUGU 1177 AD-73946 A-148037 CAGCUGUCAUCAAUUAAAGdTdT 808A-148038 CUUUAAUUGAUGACAGCUGdTdT 993 CAGCUGUCAUCAAUUAAAG 1178 AD-73947A-148039 AAUUAAAGGCAAAGGCAAUdTdT 809 A-148040 AUUGCCUUUGCCUUUAAUUdTdT994 AAUUAAAGGCAAAGGCAAU 1179 AD-73948 A-148041 AAGGCAAUCGAAUCUAAAAdTdT810 A-148042 UUUUAGAUUCGAUUGCCUUdTdT 995 AAGGCAAUCGAAUCUAAAA 1180

Example 5-In Vitro Screening

Cell Culture and Plasmids/Transfections for Dual-Glo® Assay:

HeLa cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus 0.1μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. Forty μl of Dulbecco'sModified Eagle Medium (Life Tech) containing ˜5×10³ cells were thenadded to the siRNA mixture. Cells were incubated for 24 hours prior toRNA purification. Single dose experiments were performed at 10 nM and0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl ofLysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μ1 of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNaseinhibitor and 6.6 μl of H₂O per reaction was added to RNA isolatedabove. Plates were sealed, mixed, and incubated on an electromagneticshaker for 10 minutes at room temperature, followed by 2 hours at 37° C.

Real Time PCR

Two μ1 of cDNA were added to a master mix containing 0.5 μl of HumanGAPDH TaqMan Probe (4326317E), 0.5 μl IGF-1 human probe (Hs01547656_m1)and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) perwell in a 384 well plates (Roche cat #04887301001). Real time PCR wasdone in a LightCycler480 Real Time PCR system (Roche). Each duplex wastested in duplicate and data were normalized to cells transfected with anon-targeting control siRNA.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with a non-targeting control siRNA.

TABLE 9 Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAsSense Range SEQ Antisense Range SEQ Duplex Oligo in SEQ ID Oligo in SEQID Name Name Sense sequence ID No.: 11 NO Name Antisense equenceID No.: 11 NO AD-66716 A-133440 GCUGCUUCCGGAGCUGUGAUA 548-568 1181A-133441 UAUCACAGCUCCGGAAGCAGCAC 546-568 1247 AD-66717 A-133442UCUGCGGGGCUGAGCUGGUGA 422-442 1182 A-133443 UCACCAGCUCAGCCCCGCAGAGC420-442 1248 AD-66718 A-133444 CCUGCUCACCUUCACCAGCUA 378-398 1183A-133445 UAGCUGGUGAAGGUGAGCAGGCA 376-398 1249 AD-66719 A-133446GUGGAGACAGGGGCUUUUAUU 461-481 1184 A-133447 AAUAAAAGCCCCUGUCUCCACAC459-481 1250 AD-66720 A-133448 UGGAGACAGGGGCUUUUAUUU 462-482 1185A-133449 AAAUAAAAGCCCCUGUCUCCACA 460-482 1251 AD-66721 A-133450GAGACAGGGGCUUUUAUUUCA 464-484 1186 A-133451 UGAAAUAAAAGCCCCUGUCUCCA462-484 1252 AD-66722 A-133452 CAUGUCCUCCUCGCAUCUCUU 342-362 1187A-133453 AAGAGAUGCGAGGAGGACAUGGU 340-362 1253 AD-66723 A-133454UUUUAUUUCAACAAGCCCACA 475-495 1188 A-133455 UGUGGGCUUGUUGAAAUAAAAGC473-495 1254 AD-66724 A-133456 UGUGGAGACAGGGGCUUUUAU 460-480 1189A-133457 AUAAAAGCCCCUGUCUCCACACA 458-480 1255 AD-66725 A-133458UGGAUGAGUGCUGCUUCCGGA 539-559 1190 A-133459 UCCGGAAGCAGCACUCAUCCACG537-559 1256 AD-66726 A-133460 UCGUGUGUGGAGACAGGGGCU 455-475 1191A-133461 AGCCCCUGUCUCCACACACGAAC 453-475 1257 AD-66727 A-133462GAUGUAUUGCGCACCCCUCAA 582-602 1192 A-133463 UUGAGGGGUGCGCAAUACAUCUC580-602 1258 AD-66728 A-133464 UUCAGUUCGUGUGUGGAGACA 449-469 1193A-133465 UGUCUCCACACACGAACUGAAGA 447-469 1259 AD-66729 A-133466CUCCUCGCAUCUCUUCUACCU 348-368 1194 A-133467 AGGUAGAAGAGAUGCGAGGAGGA346-368 1260 AD-66730 A-133468 AGAUGUAUUGCGCACCCCUCA 581-601 1195A-133469 UGAGGGGUGCGCAAUACAUCUCC 579-601 1261 AD-66731 A-133470GCCACACCGACAUGCCCAAGA 638-658 1196 A-133471 UCUUGGGCAUGUCGGUGUGGCGC636-658 1262 AD-66732 A-133472 GGAGAUGUAUUGCGCACCCCU 579-599 1197A-133473 AGGGGUGCGCAAUACAUCUCCAG 577-599 1263 AD-66733 A-133474UUCAACAAGCCCACAGGGUAU 481-501 1198 A-133475 AUACCCUGUGGGCUUGUUGAAAU479-501 1264 AD-66734 A-133476 UGCCCAGCGCCACACCGACAU 630-650 1199A-133478 AUGUCGGUGUGGCGCUGGGCACG 628-650 1265 AD-66735 A-133480GCAUCGUGGAUGAGUGCUGCU 533-553 1200 A-133482 AGCAGCACUCAUCCACGAUGCCU531-553 1266 AD-66736 A-133484 GGAGACAGGGGCUUUUAUUUA 463-483 1201A-133486 UAAAUAAAAGCCCCUGUCUCCAC 461-483 1267 AD-66737 A-133488GGGCUUUUAUUUCAACAAGCA 471-491 1202 A-133490 UGCUUGUUGAAAUAAAAGCCCCU469-491 1268 AD-66738 A-133492 UCGUGGAUGAGUGCUGCUUCA 536-556 1203A-133494 UGAAGCAGCACUCAUCCACGAUG 534-556 1269 AD-66739 A-133496UCCUCGCAUCUCUUCUACCUA 349-369 1204 A-133498 UAGGUAGAAGAGAUGCGAGGAGG347-369 1270 AD-66740 A-133500 GGGGCUUUUAUUUCAACAAGA 470-490 1205A-133502 UCUUGUUGAAAUAAAAGCCCCUG 468-490 1271 AD-66741 A-133504UUUAUUUCAACAAGCCCACAA 476-496 1206 A-133506 UUGUGGGCUUGUUGAAAUAAAAG474-496 1272 AD-66742 A-133508 GCUGGAGAUGUAUUGCGCACA 576-596 1207A-133510 UGUGCGCAAUACAUCUCCAGCCU 574-596 1273 AD-66743 A-133512GGGUAUGGCUCCAGCAGUCGA 496-516 1208 A-133513 UCGACUGCUGGAGCCAUACCCUG494-516 1274 AD-66744 A-133514 CUGGAGAUGUAUUGCGCACCA 577-597 1209A-133515 UGGUGCGCAAUACAUCUCCAGCC 575-597 1275 AD-66745 A-133516GAAGAUGCACACCAUGUCCUA 330-350 1210 A-133517 UAGGACAUGGUGUGCAUCUUCAC328-350 1276 AD-66746 A-133477 GAUGCUCUUCAGUUCGUGUGU 442-462 1211A-133479 ACACACGAACUGAAGAGCAUCCA 440-462 1277 AD-66747 A-133481UGGAUGCUCUUCAGUUCGUGU 440-460 1212 A-133483 ACACGAACUGAAGAGCAUCCACC438-460 1278 AD-66748 A-133485 GGUGGAUGCUCUUCAGUUCGU 438-458 1213A-133487 ACGAACUGAAGAGCAUCCACCAG 436-458 1279 AD-66749 A-133489UGAGCUGGUGGAUGCUCUUCA 432-452 1214 A-133491 UGAAGAGCAUCCACCAGCUCAGC430-452 1280 AD-66750 A-133493 GCUGGUGGAUGCUCUUCAGUU 435-455 1215A-133495 AACUGAAGAGCAUCCACCAGCUC 433-455 1281 AD-66751 A-133497GGAUGCUCUUCAGUUCGUGUA 441-461 1216 A-133499 UACACGAACUGAAGAGCAUCCAC439-461 1282 AD-66752 A-133501 CUGGUGGAUGCUCUUCAGUUA 436-456 1217A-133503 UAACUGAAGAGCAUCCACCAGCU 434-456 1283 AD-66753 A-133505GGCUGAGCUGGUGGAUGCUCU 429-449 1218 A-133507 AGAGCAUCCACCAGCUCAGCCCC427-449 1284 AD-66754 A-133509 CUGAGCUGGUGGAUGCUCUUA 431-451 1219A-133511 UAAGAGCAUCCACCAGCUCAGCC 429-451 1285 AD-66755 A-133518CAUUGUGGAUGAGUGUUGCUU 534-554 1220 A-133519 AAGCAACACUCAUCCACAAUGCC532-554 1286 AD-66756 A-133520 AGAUACACAUCAUGUCGUCUU * 1221 A-133521AAGACGACAUGAUGUGUAUCUUU * 1287 AD-66757 A-133522 UGGAUGAGUGUUGCUUCCGGA539-559 1222 A-133523 UCCGGAAGCAACACUCAUCCACA 537-559 1288 AD-66758A-133524 UGUUGCUUCCGGAGCUGUGAU 547-567 1223 A-133525AUCACAGCUCCGGAAGCAACACU 545-567 1289 AD-66759 A-133526GCUUUUACUUCAACAAGCCCA 473-493 1224 A-133527 UGGGCUUGUUGAAGUAAAAGCCC471-493 1290 AD-66760 A-133528 AUGAGUGUUGCUUCCGGAGCU 542-562 1225A-133529 AGCUCCGGAAGCAACACUCAUCC 540-562 1291 AD-66761 A-133530CACACUGACAUGCCCAAGACU 640-660 1226 A-133531 AGUCUUGGGCAUGUCAGUGUGGC638-660 1292 AD-66762 A-133532 GCUAUGGCUCCAGCAUUCGGA 497-517 1227A-133533 UCCGAAUGCUGGAGCCAUAGCCU 495-517 1293 AD-66763 A-133534AAGAUACACAUCAUGUCGUCU * 1228 A-133535 AGACGACAUGAUGUGUAUCUUUA * 1294AD-66764 A-133536 UUGCUUCCGGAGCUGUGAUCU 549-569 1229 A-133537AGAUCACAGCUCCGGAAGCAACA 547-569 1295 AD-66765 A-133538UCCGGAGCUGUGAUCUGAGGA 554-574 1230 A-133539 UCCUCAGAUCACAGCUCCGGAAG552-574 1296 AD-66766 A-133540 GUGGAUGAGUGUUGCUUCCGA 538-558 1231A-133541 UCGGAAGCAACACUCAUCCACAA 536-558 1297 AD-66767 A-133542UACACAUCAUGUCGUCUUCAA * 1232 A-133543 UUGAAGACGACAUGAUGUGUAUC * 1298AD-66768 A-133544 AAAGAUACACAUCAUGUCGUA * 1233 A-133545UACGACAUGAUGUGUAUCUUUAU * 1299 AD-66769 A-133546 GGCUAUGGCUCCAGCAUUCGA496-516 1234 A-133547 UCGAAUGCUGGAGCCAUAGCCUG 494-516 1300 AD-66770A-133548 ACACUGACAUGCCCAAGACUA 641-661 1235 A-133549UAGUCUUGGGCAUGUCAGUGUGG 639-661 1301 AD-66771 A-133550AGUGUUGCUUCCGGAGCUGUA 545-565 1236 A-133551 UACAGCUCCGGAAGCAACACUCA543-565 1302 AD-66772 A-133552 GAGACCCUUUGCGGGGCUGAA * 1237 A-133553UUCAGCCCCGCAAAGGGUCUCUG * 1303 AD-66773 A-133554 ACUGACAUGCCCAAGACUCAA643-663 1238 A-133555 UUGAGUCUUGGGCAUGUCAGUGU 641-663 1304 AD-66774A-133556 GAUACACAUCAUGUCGUCUUA * 1239 A-133557 UAAGACGACAUGAUGUGUAUCUU *1305 AD-66775 A-133558 AAGCCCACAGGCUAUGGCUCA 487-507 1240 A-133559UGAGCCAUAGCCUGUGGGCUUGU 485-507 1306 Sense Range SEQ Antisense Range SEQDuplex Oligo in SEQ ID Oligo in SEQ ID Name Name Sense sequenceID No.: 17 NO Name Antisense equence ID No.: 17 NO AD-66756 A-133520AGAUACACAUCAUGUCGUCUU 366-368 1241 A-133521 AAGACGACAUGAUGUGUAUCUUU364-368 1307 AD-66763 A-133534 AAGAUACACAUCAUGUCGUCU 365-385 1242A-133535 AGACGACAUGAUGUGUAUCUUUA 363-385 1308 AD-66767 A-133542UACACAUCAUGUCGUCUUCAA 369-389 1243 A-133543 UUGAAGACGACAUGAUGUGUAUC367-389 1309 AD-66768 A-133544 AAAGAUACACAUCAUGUCGUA 364-384 1244A-133545 UACGACAUGAUGUGUAUCUUUAU 362-384 1310 AD-66772 A-133552GAGACCCUUUGCGGGGCUGAA 449-469 1245 A-133553 UUCAGCCCCGCAAAGGGUCUCUG447-469 1311 AD-66774 A-133556 GAUACACAUCAUGUCGUCUUA 367-387 1246A-133557 UAAGACGACAUGAUGUGUAUCUU 365-387 1312 *Targeting sequence inNM_010512 (SEQ ID NO: 7).

TABLE 10 IGF-1 Screen in HeLa cells Each duplex was tested in duplicateand data were normalized to cells transfected with a non-targetingcontrol siRNA AD-1955. Duplex ID 10 nM Avg STDEV 0.1 nM Avg STDEVAD-66716 58.4 3.1 90.3 24.0 AD-66717 82.2 1.6 81.1 10.3 AD-66718 62.77.0 76.4 10.1 AD-66719 51.0 6.7 83.2 8.1 AD-66720 30.3 0.9 74.9 15.7AD-66721 58.0 8.5 82.5 23.9 AD-66722 4.7 1.0 29.2 9.0 AD-66723 70.5 7.385.4 11.3 AD-66724 32.4 8.0 82.7 9.7 AD-66725 22.6 6.4 72.8 12.1AD-66726 32.8 2.1 76.4 25.0 AD-66727 53.5 1.1 83.1 6.5 AD-66728 59.111.5 91.5 15.2 AD-66729 27.9 4.2 75.3 27.4 AD-66730 79.1 12.7 87.8 18.4AD-66731 86.4 15.9 97.0 25.4 AD-66732 81.0 8.3 80.9 15.0 AD-66733 8.73.7 43.7 1.9 AD-66734 65.4 3.2 83.7 15.5 AD-66735 62.0 6.7 82.6 8.1AD-66736 71.9 4.5 83.5 23.4 AD-66737 68.7 4.0 92.0 14.4 AD-66738 19.23.3 79.4 14.3 AD-66739 10.6 2.7 61.8 23.6 AD-66740 23.2 4.4 68.2 14.6AD-66741 83.6 0.5 76.5 14.2 AD-66742 73.1 2.2 86.2 10.1 AD-66743 58.91.8 88.1 11.2 AD-66744 53.9 2.1 96.8 7.6 AD-66745 28.3 5.5 76.8 7.9AD-66746 6.3 0.7 50.1 3.9 AD-66747 8.5 2.8 50.0 0.0 AD-66748 6.2 1.334.8 4.1 AD-66749 30.0 0.5 92.4 3.2 AD-66750 27.6 0.7 74.8 1.5 AD-6675150.1 0.3 89.3 15.2 AD-66752 9.6 1.3 55.5 12.4 AD-66753 54.6 2.1 89.0 0.9AD-66754 78.6 16.8 104.3 2.0 AD-66755 46.8 4.8 103.4 18.6 AD-66756 86.32.5 95.9 18.2 AD-66757 69.1 0.7 103.0 5.0 AD-66758 67.5 3.6 86.5 2.5AD-66759 106.5 16.2 91.4 20.4 AD-66760 54.2 1.6 86.8 0.4 AD-66761 40.83.8 92.2 7.2 AD-66762 96.8 7.1 100.6 8.4 AD-66763 81.2 11.4 92.1 0.0AD-66764 86.0 2.2 101.2 12.9 AD-66765 100.9 13.7 93.2 25.3 AD-66766 36.63.9 78.5 15.7 AD-66767 124.0 12.2 89.9 1.3 AD-66768 113.9 15.1 92.7 15.4AD-66769 92.0 7.1 93.6 8.2 AD-66770 79.7 3.6 98.7 1.9 AD-66771 60.6 12.197.6 10.5 AD-66772 95.5 7.0 95.9 9.9 AD-66773 61.3 3.9 90.7 7.5 AD-6677495.6 8.9 81.9 21.8 AD-66775 113.1 13.9 99.5 6.8 AD-1955 100.0 8.0

TABLE 11 Modified Sense and Antisense Sequences of IGF-1 Sense SEQAntisense SEQ Duplex Oligo ID Oligo ID Name Name Modified Sense SequenceNO Name Modified Antisense Sequence NO: AD-66716 A-133440GfscsUfgCfuUfcCfGfGfaGfcUfgUfgAfuAfL96 1313 A-133441usAfsuCfaCfaGfcUfccgGfaAfgCfaGfcsasc 1373 AD-66717 A-133442UfscsUfgCfgGfgGfCfUfgAfgCfuGfgUfgAfL96 1314 A-133443usCfsaCfcAfgCfuCfagcCfcCfgCfaGfasgsc 1374 AD-66718 A-133444CfscsUfgCfuCfaCfCfUfuCfaCfcAfgCfuAfL96 1315 A-133445usAfsgCfuGfgUfgAfaggUfgAfgCfaGfgscsa 1375 AD-66719 A-133446GfsusGfgAfgAfcAfGfGfgGfcUfuUfuAfuUfL96 1316 A-133447asAfsuAfaAfaGfcCfccuGfuCfuCfcAfcsasc 1376 AD-66720 A-133448UfsgsGfaGfaCfaGfGfGfgCfuUfuUfaUfuUfL96 1317 A-133449asAfsaUfaAfaAfgCfcccUfgUfcUfcCfascsa 1377 AD-66721 A-133450GfsasGfaCfaGfgGfGfCfuUfuUfaUfuUfcAfL96 1318 A-133451usGfsaAfaUfaAfaAfgccCfcUfgUfcUfcscsa 1378 AD-66722 A-133452CfsasUfgUfcCfuCfCfUfcGfcAfuCfuCfuUfL96 1319 A-133453asAfsgAfgAfuGfcGfaggAfgGfaCfaUfgsgsu 1379 AD-66723 A-133454UfsusUfuAfuUfuCfAfAfcAfaGfcCfcAfcAfL96 1320 A-133455usGfsuGfgGfcUfuGfuugAfaAfuAfaAfasgsc 1380 AD-66724 A-133456UfsgsUfgGfaGfaCfAfGfgGfgCfuUfuUfaUfL96 1321 A-133457asUfsaAfaAfgCfcCfcugUfcUfcCfaCfascsa 1381 AD-66725 A-133458UfsgsGfaUfgAfgUfGfCfuGfcUfuCfcGfgAfL96 1322 A-133459usCfscGfgAfaGfcAfgcaCfuCfaUfcCfascsg 1382 AD-66726 A-133460UfscsGfuGfuGfuGfGfAfgAfcAfgGfgGfcUfL96 1323 A-133461asGfscCfcCfuGfuCfuccAfcAfcAfcGfasasc 1383 AD-66727 A-133462GfsasUfgUfaUfuGfCfGfcAfcCfcCfuCfaAfL96 1324 A-133463usUfsgAfgGfgGfuGfcgcAfaUfaCfaUfcsusc 1384 AD-66728 A-133464UfsusCfaGfuUfcGfUfGfuGfuGfgAfgAfcAfL96 1325 A-133465usGfsuCfuCfcAfcAfcacGfaAfcUfgAfasgsa 1385 AD-66729 A-133466CfsusCfcUfcGfcAfUfCfuCfuUfcUfaCfcUfL96 1326 A-133467asGfsgUfaGfaAfgAfgauGfcGfaGfgAfgsgsa 1386 AD-66730 A-133468AfsgsAfuGfuAfuUfGfCfgCfaCfcCfcUfcAfL96 1327 A-133469usGfsaGfgGfgUfgCfgcaAfuAfcAfuCfuscsc 1387 AD-66731 A-133470GfscsCfaCfaCfcGfAfCfaUfgCfcCfaAfgAfL96 1328 A-133471usCfsuUfgGfgCfaUfgucGfgUfgUfgGfcsgsc 1388 AD-66732 A-133472GfsgsAfgAfuGfuAfUfUfgCfgCfaCfcCfcUfL96 1329 A-133473asGfsgGfgUfgCfgCfaauAfcAfuCfuCfcsasg 1389 AD-66733 A-133474UfsusCfaAfcAfaGfCfCfcAfcAfgGfgUfaUfL96 1330 A-133475asUfsaCfcCfuGfuGfggcUfuGfuUfgAfasasu 1390 AD-66734 A-133476UfsgsCfcCfaGfcGfCfCfaCfaCfcGfaCfaUfL96 1331 A-133478asUfsgUfcGfgUfgUfggeGfcUfgGfgCfascsg 1391 AD-66735 A-133480GfscsAfuCfgUfgGfAfUfgAfgUfgCfuGfcUfL96 1332 A-133482asGfscAfgCfaCfuCfaucCfaCfgAfuGfcscsu 1392 AD-66736 A-133484GfsgsAfgAfcAfgGfGfGfcUfuUfuAfuUfuAfL96 1333 A-133486usAfsaAfuAfaAfaGfcccCfuGfuCfuCfcsasc 1393 AD-66737 A-133488GfsgsGfcUfuUfuAfUfUfuCfaAfcAfaGfcAfL96 1334 A-133490usGfscUfuGfuUfgAfaauAfaAfaGfcCfcscsu 1394 AD-66738 A-133492UfscsGfuGfgAfuGfAfGfuGfcUfgCfuUfcAfL96 1335 A-133494usGfsaAfgCfaGfcAfcucAfuCfcAfcGfasusg 1395 AD-66739 A-133496UfscsCfuCfgCfaUfCfUfcUfuCfuAfcCfuAfL96 1336 A-133498usAfsgGfuAfgAfaGfagaUfgCfgAfgGfasgsg 1396 AD-66740 A-133500GfsgsGfgCfuUfuUfAfUfuUfcAfaCfaAfgAfL96 1337 A-133502usCfsuUfgUfuGfaAfauaAfaAfgCfcCfcsusg 1397 AD-66741 A-133504UfsusUfaUfuUfcAfAfCfaAfgCfcCfaCfaAfL96 1338 A-133506usUfsgUfgGfgCfuUfguuGfaAfaUfaAfasasg 1398 AD-66742 A-133508GfscsUfgGfaGfaUfGfUfaUfuGfcGfcAfcAfL96 1339 A-133510usGfsuGfcGfcAfaUfacaUfcUfcCfaGfcscsu 1399 AD-66743 A-133512GfsgsGfuAfuGfgCfUfCfcAfgCfaGfuCfgAfL96 1340 A-133513usCfsgAfcUfgCfuGfgagCfcAfuAfcCfcsusg 1400 AD-66744 A-133514CfsusGfgAfgAfuGfUfAfuUfgCfgCfaCfcAfL96 1341 A-133515usGfsgUfgCfgCfaAfuacAfuCfuCfcAfgscsc 1401 AD-66745 A-133516GfsasAfgAfuGfcAfCfAfcCfaUfgUfcCfuAfL96 1342 A-133517usAfsgGfaCfaUfgGfuguGfcAfuCfuUfcsasc 1402 AD-66746 A-133477GfsasUfgCfuCfuUfCfAfgUfuCfgUfgUfgUfL96 1343 A-133479asCfsaCfaCfgAfaCfugaAfgAfgCfaUfcscsa 1403 AD-66747 A-133481UfsgsGfaUfgCfuCfUfUfcAfgUfuCfgUfgUfL96 1344 A-133483asCfsaCfgAfaCfuGfaagAfgCfaUfcCfascsc 1404 AD-66748 A-133485GfsgsUfgGfaUfgCfUfCfuUfcAfgUfuCfgUfL96 1345 A-133487asCfsgAfaCfuGfaAfgagCfaUfcCfaCfcsasg 1405 AD-66749 A-133489UfsgsAfgCfuGfgUfGfGfaUfgCfuCfuUfcAfL96 1346 A-133491usGfsaAfgAfgCfaUfccaCfcAfgCfuCfasgsc 1406 AD-66750 A-133493GfscsUfgGfuGfgAfUfGfcUfcUfuCfaGfuUfL96 1347 A-133495asAfscUfgAfaGfaGfcauCfcAfcCfaGfcsusc 1407 AD-66751 A-133497GfsgsAfuGfcUfcUfUfCfaGfuUfcGfuGfuAfL96 1348 A-133499usAfscAfcGfaAfcUfgaaGfaGfcAfuCfcsasc 1408 AD-66752 A-133501CfsusGfgUfgGfaUfGfCfuCfuUfcAfgUfuAfL96 1349 A-133503usAfsaCfuGfaAfgAfgcaUfcCfaCfcAfgscsu 1409 AD-66753 A-133505GfsgsCfuGfaGfcUfGfGfuGfgAfuGfcUfcUfL96 1350 A-133507asGfsaGfcAfuCfcAfccaGfcUfcAfgCfcscsc 1410 AD-66754 A-133509CfsusGfaGfcUfgGfUfGfgAfuGfcUfcUfuAfL96 1351 A-133511usAfsaGfaGfcAfuCfcacCfaGfcUfcAfgscsc 1411 AD-66755 A-133518CfsasUfuGfuGfgAfUfGfaGfuGfuUfgCfuUfL96 1352 A-133519asAfsgCfaAfcAfcUfcauCfcAfcAfaUfgscsc 1412 AD-66756 A-133520AfsgsAfuAfcAfcAfUfCfaUfgUfcGfuCfuUfL96 1353 A-133521asAfsgAfcGfaCfaUfgauGfuGfuAfuCfususu 1413 AD-66757 A-133522UfsgsGfaUfgAfgUfGfUfuGfcUfuCfcGfgAfL96 1354 A-133523usCfscGfgAfaGfcAfacaCfuCfaUfcCfascsa 1414 AD-66758 A-133524UfsgsUfuGfcUfuCfCfGfgAfgCfuGfuGfaUfL96 1355 A-133525asUfscAfcAfgCfuCfcggAfaGfcAfaCfascsu 1415 AD-66759 A-133526GfscsUfuUfuAfcUfUfCfaAfcAfaGfcCfcAfL96 1356 A-133527usGfsgGfcUfuGfuUfgaaGfuAfaAfaGfcscsc 1416 AD-66760 A-133528AfsusGfaGfuGfuUfGfCfuUfcCfgGfaGfcUfL96 1357 A-133529asGfscUfcCfgGfaAfgcaAfcAfcUfcAfuscsc 1417 AD-66761 A-133530CfsasCfaCfuGfaCfAfUfgCfcCfaAfgAfcUfL96 1358 A-133531asGfsuCfuUfgGfgCfaugUfcAfgUfgUfgsgsc 1418 AD-66762 A-133532GfscsUfaUfgGfcUfCfCfaGfcAfuUfcGfgAfL96 1359 A-133533usCfscGfaAfuGfcUfggaGfcCfaUfaGfcscsu 1419 AD-66763 A-133534AfsasGfaUfaCfaCfAfUfcAfuGfuCfgUfcUfL96 1360 A-133535asGfsaCfgAfcAfuGfaugUfgUfaUfcUfususa 1420 AD-66764 A-133536UfsusGfcUfuCfcGfGfAfgCfuGfuGfaUfcUfL96 1361 A-133537asGfsaUfcAfcAfgCfuccGfgAfaGfcAfascsa 1421 AD-66765 A-133538UfscsCfgGfaGfcUfGfUfgAfuCfuGfaGfgAfL96 1362 A-133539usCfscUfcAfgAfuCfacaGfcUfcCfgGfasasg 1422 AD-66766 A-133540GfsusGfgAfuGfaGfUfGfuUfgCfuUfcCfgAfL96 1363 A-133541usCfsgGfaAfgCfaAfcacUfcAfuCfcAfcsasa 1423 AD-66767 A-133542UfsasCfaCfaUfcAfUfGfuCfgUfcUfuCfaAfL96 1364 A-133543usUfsgAfaGfaCfgAfcauGfaUfgUfgUfasusc 1424 AD-66768 A-133544AfsasAfgAfuAfcAfCfAfuCfaUfgUfcGfuAfL96 1365 A-133545usAfscGfaCfaUfgAfuguGfuAfuCfuUfusasu 1425 AD-66769 A-133546GfsgsCfuAfuGfgCfUfCfcAfgCfaUfuCfgAfL96 1366 A-133547usCfsgAfaUfgCfuGfgagCfcAfuAfgCfcsusg 1426 AD-66770 A-133548AfscsAfcUfgAfcAfUfGfcCfcAfaGfaCfuAfL96 1367 A-133549usAfsgUfcUfuGfgGfcauGfuCfaGfuGfusgsg 1427 AD-66771 A-133550AfsgsUfgUfuGfcUfUfCfcGfgAfgCfuGfuAfL96 1368 A-133551usAfscAfgCfuCfcGfgaaGfcAfaCfaCfuscsa 1428 AD-66772 A-133552GfsasGfaCfcCfuUfUfGfcGfgGfgCfuGfaAfL96 1369 A-133553usUfscAfgCfcCfcGfcaaAfgGfgUfcUfcsusg 1429 AD-66773 A-133554AfscsUfgAfcAfuGfCfCfcAfaGfaCfuCfaAfL96 1370 A-133555usUfsgAfgUfcUfuGfggcAfuGfuCfaGfusgsu 1430 AD-66774 A-133556GfsasUfaCfaCfaUfCfAfuGfuCfgUfcUfuAfL96 1371 A-133557usAfsaGfaCfgAfcAfugaUfgUfgUfaUfcsusu 1431 AD-66775 A-133558AfsasGfcCfcAfcAfGfGfcUfaUfgGfcUfcAfL96 1372 A-133559usGfsaGfcCfaUfaGfccuGfuGfgGfcUfusgsu 1432

Example 6-Knockdown of IGF-1 Expression with an IGF-1 siRNA DecreasesExpression of IGF-1

A series of siRNAs targeting mouse IGF-1 were designed and tested forthe ability to knockdown expression of IGF-1 mRNA in 6-8 week oldC57B1/6 female mice. Duplexes were selected for further optimizationusing chemical modifications. Analysis for IGF-1 knockdown in mice usingthe same assay identified the AD-68112 duplex (sense sequencegsasuacaCfaUfCfAfugucgucuuaL96 (SEQ ID NO:1433); antisense sequenceusAfsagaCfgAfCfaugaUfgUfguaucsusu (SEQ ID NO: 1434), based on thesequence of the AD-66774 duplex, for use in further studies.

A single 3 mg/kg or 10 mg/kg dose of AD-68112; or PBS control, wasadministered subcutaneously on day 0 to 6-8 week old C57B1/6 female mice(n=3 per group). On days 7, 14, and 21 the mice were sacrificed toassess knockdown of IGF-1 mRNA in liver by qPCR.

AD-68112 was found to be effective in decreasing expression of IGF-1mRNA. The results are shown in the table below. Results are expressed asmRNA levels relative to control.

Dose Day 7 Day 14 Day 21  3 mg/kg 7.4 17.8 33.3 10 mg/kg 3.4 7.4 12.7

In a separate study, a decrease in serum IGF-1 was observed in 6-8 weekold C57B1/6 female mice (n=3) response to treatment with AD-68112 in asingle dose of 3 mg/kg. Specifically, AD-68112 decreased the serum IGF-1protein level to about 17%, 70%, and 55% on days 7, 14, and 21,respectively.

Further, in a dose-response study, AD-68112 was demonstrated to beeffective in knocking down the expression of IGF-1 mRNA in the liver ina dose-response manner. Specifically, C57B1/6 female mice, 6-8 weeks ofage (n=3 per group) were administered a single 0.3 mg/kg, 1 mg/kg, 3mg/kg, or 10 mg/kg AD-68112 at; or a PBS control. IGF-1 serum proteinlevel reduction was observed in a dose response manner.

Example 7-Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1siRNAs Alone and in Combination

A series of siRNAs targeted each to mouse IGFALS (AD-66807, sensesequence, ascsagauGfaGfCfUfcagcgucuuuL96, SEQ ID NO: 1435; and antisensesequence asAfsagaCfgCfUfgagcUfcAfucugusgsu, SEQ ID NO:1436) and mouseIGF-1 (AD-68112) were tested for the ability to knockdown expression ofIGFALS and IGF-1 mRNA and protein expression in 6-8 week old C57B1/6female mice, either alone or in combination.

Weekly 3 mg/kg doses of AD-66807 and AD-68112, either alone or incombination; or PBS control, were administered to 4 week old C57B1/6female mice (n=8 per group) subcutaneously starting at day 0 for 8weeks. On day 58 or 59, the mice were sacrificed to assess knockdown ofIGFALS and IGF-1 mRNA in liver by qPCR. Serum IGFALS levels were assayedby western blot and serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasingexpression of IGFALS and IGF-1 mRNA and protein. The results are shownin the tables below. RNA and protein levels are expressed as levelsrelative to control.

IGFALS and IGF-1 mRNA Levels Relative to Control

Treatment IGFALS IGF-1 Control (PBS) 1.16 1.05 SiRNA-IGFALS (3 mg/kg)0.11 1.10 SiRNA-IGF-1 (3 mg/kg) 0.70 0.03 SiRNA-ALS (3 mg/kg) + IGF-1 (3mg/kg) 0.09 0.05IGFALS and IGF-1 Protein Levels Relative to Control.

Treatment IGFALS IGF-1 Control (PBS) 1.0 1.0 SiRNA-IGFALS (3 mg/kg) 0.030.26 SiRNA-IGF-1 (3 mg/kg) 0.61 0.13 SiRNA-ALS (3 mg/kg) + IGF-1 (3mg/kg) 0.01 0.05

A dose response study was performed. AD-66807 and AD-68112 weresubcutaneously administered to 6-8 week old C57Bl/6 female mice (n=3 pergroup) at 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg, either alone or incombination; or PBS control starting at day 0. On day 14, the mice weresacrificed to assess knockdown of IGFALS and IGF-1 mRNA in liver byqPCR. Serum IGF-1 levels were assayed by ELISA.

AD-66807 and AD-68112 were found to be effective in decreasingexpression of serum IGF-1 protein. The results are shown in the tablebelow. Protein levels are expressed as levels relative to control.

Serum IGF-1 Protein Levels Relative to Control at Day 7.

3.0 10.0 Treatment 0 mg/kg 0.3 mg/kg 1.0 mg/kg mg/kg mg/kg SiRNA-IGFALS100 0.81 0.54 0.37 0.31 SiRNA-IGF-1 100 1.13 0.83 0.52 0.49SiRNA-IGFALS + 100 0.61 0.34 0.14 0.06 SiRNA-IGF-1

Example 8-Knockdown of IGFALS and IGF-1 Expression with IGFALS and IGF-1siRNAs Alone and in Combination in a Transgenic Mouse Expressing BovineGrowth Hormone

Similar knockdown of serum IGFALS and IGF-1 levels were observed in atransgenic mouse that constitutively expresses bovine growth hormone andrecapitulates some of the features of acromegaly (Olsson et al, Am JPhysiol Endocrinol Metab. 2003; 285:E504-11). Specifically, AD-66807 andAD-68112 were demonstrated to decrease serum levels of IGF-1 protein. Atleast a trend in decreased weight gain was observed in male mice treatedwith either AD-66807 or AD-68112 alone, and in female mice treated witha combination of AD-66807 and AD-68112.

Example 9-IGFALS Transcripts, siRNA Design, and siRNA Screening

A set of siRNAs targeting the human IGFALS, “insulin like growth factorbinding protein acid labile subunit” (human: NCBI refseqID NM_004970(SEQ ID NO: 1); NCBI GeneID: 3483), as well as toxicology-species IGFALSorthologs (cynomolgus monkey: XM_005590898) were designed using custom Rand Python scripts. The human NM_004970 REFSEQ mRNA, version 2, has alength of 2168 bases.

The rationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 10through the end was determined with a linear model derived the directmeasure of mRNA knockdown from more than 20,000 distinct siRNA designstargeting a large number of vertebrate genes. Subsets of the IGFALSsiRNAs were designed with perfect or near-perfect matches between humanand cynomolgus monkey. For each strand of the siRNA, a custom Pythonscript was used in a brute force search to measure the number andpositions of mismatches between the siRNA and all potential alignmentsin the target species transcriptome. Extra weight was given tomismatches in the seed region, defined here as positions 2-9 of theantisense oligonucleotide, as well the cleavage site of the siRNA,defined here as positions 10-11 of the antisense oligonucleotide. Therelative weight of the mismatches was 2.8; 1.2:1 for seed mismatches,cleavage site, and other positions up through antisense position 19.Mismatches in the first position were ignored. A specificity score wascalculated for each strand by summing the value of each weightedmismatch. Preference was given to siRNAs whose antisense score in humanwas >=2.2 and predicted efficacy was >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Human IGFALS(NM_004970 (SEQ ID NO:1) was cloned into the psicheck2 vector togenerate the Dual-Glo® Luciferase construct. The Dual-luciferase plasmidwas co-transfected with siRNA into 5000 cells using LipofectamineRNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a384 well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmidvector and siRNA in 15 μl of Opti-MEM and allowed to complex at roomtemperature for 15 minutes. The mixture was then added to the cellsresuspended in 35 ul of fresh complete media. Cells were incubated for48 hours before luciferase was measured. Screens were performed at 10 nMand 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly(transfection control) and Renilla (fused to IGFALS target sequence in3′ UTR) luciferase were measured. First, media was removed from cells.Then Firefly luciferase activity was measured by adding 20 ul ofDual-Glo® Luciferase Reagent mixed with 20 ul of complete media to eachwell. The mixture was incubated at room temperature for 30 minutesbefore luminescense (500 nm) was measured on a Spectramax (MolecularDevices) to detect the Firefly luciferase signal. Renilla luciferaseactivity was measured by adding 20 ul of room temperature of Dual-Glo®Stop & Glo® Reagent to each well and the plates were incubated for 20minutes before luminescence was again measured to determine the Renillaluciferase signal. The Dual-Glo® Stop & Glo® Reagent quenched thefirefly luciferase signal and sustained luminescence for the Renillaluciferase reaction. siRNA activity was determined by normalizing theRenilla (IGFALS) signal to the Firefly (control) signal within eachwell. The magnitude of siRNA activity was then assessed relative tocells that were transfected with the same vector but were not treatedwith siRNA or were treated with a non-targeting siRNA. All transfectionswere done in quadruplicates.

TABLE 12Unmodified Sense and Antisense Strand Sequences of IGFALS dsRNAs SensePosition SEQ Antisense Position SEQ Duplex Oligo in NM_ ID Oligo in NM_ID Name Name Sense Sequence 004970.2 NO Name Antisense Sequence 004970.2NO AD-76171 A-152158 CAAUUAAAGGCAAAGGCAAUA 2058-2078 1437 A-152159UAUUGCCUUUGCCUUUAAUUGAU 2056-2078 1484 AD-76172 A-152160ACACCUACAACAACAUCACCA 1808-1828 1438 A-152161 UGGUGAUGUUGUUGUAGGUGUAC1806-1828 1485 AD-76173 A-152162 UACACCUACAACAACAUCACA 1807-1827 1439A-152163 UGUGAUGUUGUUGUAGGUGUACG 1805-1827 1486 AD-76174 A-152164CAUCAAGGCAAACGUGUUCGA  777-797 1440 A-152165 UCGAACACGUUUGCCUUGAUGGC 775-797 1487 AD-76175 A-152166 CACCUACAACAACAUCACCUA 1809-1829 1441A-152167 UAGGUGAUGUUGUUGUAGGUGUA 1807-1829 1488 AD-76176 A-152168CGUACACCUACAACAACAUCA 1805-1825 1442 A-152169 UGAUGUUGUUGUAGGUGUACGCG1803-1825 1489 AD-76177 A-152170 GUACACCUACAACAACAUCAA 1806-1826 1443A-152171 UUGAUGUUGUUGUAGGUGUACGC 1804-1826 1490 AD-76178 A-152172GACUCUUCCUCAAGGACAACA 1322-1342 1444 A-152173 UGUUGUCCUUGAGGAAGAGUCGG1320-1342 1491 AD-76179 A-152174 AGCCUUCCAGAACCUCUCCAA  357-377 1445A-152175 UUGGAGAGGUUCUGGAAGGCUGC  355-377 1492 AD-76180 A-152176UCACGCUAGACCACAACCAGA 1109-1129 1446 A-152177 UCUGGUUGUGGUCUAGCGUGAGC1107-1129 1493 AD-76181 A-152178 UGGACGUCUCGCACAACCGCA 1541-1561 1447A-152179 UGCGGUUGUGCGAGACGUCCAGC 1539-1561 1494 AD-76182 A-152180ACCUGGACCGCAACCUCAUCA  824-844 1448 A-152181 UGAUGAGGUUGCGGUCCAGGUAG 822-844 1495 AD-76183 A-152182 UGGACCUGUCCCACAACCGCA  893-913 1449A-152183 UGCGGUUGUGGGACAGGUCCAGC  891-913 1496 AD-76184 A-152184UGCGGCUGUCCCACAACGCCA  965-985 1450 A-152185 UGGCGUUGUGGGACAGCCGCAGC 963-985 1497 AD-76185 A-152186 CGCUCGGCCUCAGCAACAACA  530-550 1451A-152187 UGUUGUUGCUGAGGCCGAGCGAG  528-550 1498 AD-76186 A-152188CCUCAGGAACAACUCACUGCA 1617-1637 1452 A-152189 UGCAGUGAGUUGUUCCUGAGGCU1615-1637 1499 AD-76187 A-152190 CUGCGGACCUUCACGCCGCAA 1633-1653 1453A-152191 UUGCGGCGUGAAGGUCCGCAGUG 1631-1653 1500 AD-76188 A-152192CCUCUCUGGGAACUGUCUCCA 1185-1205 1454 A-152193 UGGAGACAGUUCCCAGAGAGGUU1183-1205 1501 AD-76189 A-152194 GCUCUCCCGCAACCGCCUGGA 1473-1493 1455A-152195 UCCAGGCGGUUGCGGGAGAGCAG 1471-1493 1502 AD-76190 A-152196CGUCUCGCACAACCGCCUGGA 1545-1565 1456 A-152197 UCCAGGCGGUUGUGCGAGACGUC1543-1565 1503 AD-76191 A-152198 CACGCUAGACCACAACCAGCA 1110-1130 1457A-152199 UGCUGGUUGUGGUCUAGCGUGAG 1108-1130 1504 AD-76192 A-152200UGCUCUCCCGCAACCGCCUGA 1472-1492 1458 A-152201 UCAGGCGGUUGCGGGAGAGCAGC1470-1492 1505 AD-76193 A-152202 ACCUCAGCCUCAGGAACAACA 1610-1630 1459A-152203 UGUUGUUCCUGAGGCUGAGGUAG 1608-1630 1506 AD-76194 A-152204CACCUUCAAGGACCUGCACUA 1005-1025 1460 A-152205 UAGUGCAGGUCCUUGAAGGUGCG1003-1025 1507 AD-76195 A-152206 GGCCUCGUGGGCAUUGAGGAA 1342-1362 1461A-152207 UUCCUCAAUGCCCACGAGGCCGU 1340-1362 1508 AD-76196 A-152208ACCUUCAAGGACCUGCACUUA 1006-1026 1462 A-152209 UAAGUGCAGGUCCUUGAAGGUGC1004-1026 1509 AD-76197 A-152210 GGCCUUCUGGCUGGACGUCUA 1530-1550 1463A-152211 UAGACGUCCAGCCAGAAGGCCCG 1528-1550 1510 AD-76198 A-152212GGCAUUGAGGAGCAGAGCCUA 1351-1371 1464 A-152213 UAGGCUCUGCUCCUCAAUGCCCA1349-1371 1511 AD-76199 A-152214 GCCUUCCAGAACCUCUCCAGA  358-378 1465A-152215 UCUGGAGAGGUUCUGGAAGGCUG  356-378 1512 AD-76200 A-152216GCGGUCAUGAACCUCUCUGGA 1174-1194 1466 A-152217 UCCAGAGAGGUUCAUGACCGCCA1172-1194 1513 AD-76201 A-152218 AGGCUGGAGGACGGGCUCUUA  559-579 1467A-152219 UAAGAGCCCGUCCUCCAGCCUGC  557-579 1514 AD-76202 A-152220CUCUACCUGGACCGCAACCUA  820-840 1468 A-152221 UAGGUUGCGGUCCAGGUAGAGUU 818-840 1515 AD-76203 A-152222 GCGGAUGAGCUCAGCGUCUUA  238-258 1469A-152223 UAAGACGCUGAGCUCAUCCGCGU  236-258 1516 AD-76204 A-152224CCGUCUGAGCAGGCUGGAGGA  549-569 1470 A-152225 UCCUCCAGCCUGCUCAGACGGUU 547-569 1517 AD-76205 A-152226 GGUCAUGAACCUCUCUGGGAA 1176-1196 1471A-152227 UUCCCAGAGAGGUUCAUGACCGC 1174-1196 1518 AD-76206 A-152228CUGCAGCUGGGCCACAACCGA 1036-1056 1472 A-152229 UCGGUUGUGGCCCAGCUGCAGCU1034-1056 1519 AD-76207 A-152230 CGGGAGCUGGACCUGAGCAGA  742-762 1473A-152231 UCUGCUCAGGUCCAGCUCCCGGA  740-762 1520 AD-76208 A-152232CCGCCUGUGUCUGCAGCUACA  209-229 1474 A-152233 UGUAGCUGCAGACACAGGCGGCC 207-229 1521 AD-76209 A-152234 UCUUCUGCAGCUCCAGGAACA  254-274 1475A-152235 UGUUCCUGGAGCUGCAGAAGACG  252-274 1522 AD-76210 A-152236CUGGCUGAGCGCAGCUUUGAA 1066-1086 1476 A-152237 UUCAAAGCUGCGCUCAGCCAGCU1064-1086 1523 AD-76211 A-152238 CUCGACCUGACCUCCAACCAA 1396-1416 1477A-152239 UUGGUUGGAGGUCAGGUCGAGCU 1394-1416 1524 AD-76212 A-152240CUCAACCUCGGCUGGAAUAGA  604-624 1478 A-152241 UCUAUUCCAGCCGAGGUUGAGGU 602-624 1525 AD-76213 A-152242 GCCUAGAGAACCUGUGCCACA  440-460 1479A-152243 UGUGGCACAGGUUCUCUAGGCCC  438-460 1526 AD-76214 A-152244CAGCUUGAGGUGCUCACGCUA 1096-1116 1480 A-152245 UAGCGUGAGCACCUCAAGCUGCC1094-1116 1527 AD-76215 A-152246 CAGGUCCUCAGUGUCCUCAGA 1961-1981 1481A-152247 UCUGAGGACACUGAGGACCUGUC 1959-1981 1528 AD-76216 A-152248GCUGCGAUGGCUGGACCUGUA  882-902 1482 A-152249 UACAGGUCCAGCCAUCGCAGCGC 880-902 1529 AD-76217 A-152250 GGCAAGCUGGAGUACCUGCUA 1453-1473 1483A-152251 UAGCAGGUACUCCAGCUUGCCCA 1451-1473 1530

TABLE 13 IGFALS in vitro 10 nM and 0.1 nM screen Duplex 10 nM 10 nM 0.1nM Position in Name AVG STD AVG 0.1 nM STD NM_004970.2 AD-76171 29.9 0.155.8 5.4 2058-2078 AD-76172 42.7 2.3 101.9 7.8 1808-1828 AD-76173 32.83.1 81.2 7.1 1807-1827 AD-76174 45.3 10.5 86.2 9.7 777-797 AD-76175 42.87.6 97.3 8.7 1809-1829 AD-76176 69 5.8 93.9 11.3 1805-1825 AD-76177 53.611 99.2 15.8 1806-1826 AD-76178 72.6 4.9 92.6 6.9 1322-1342 AD-7617980.1 8.2 113.3 8 357-377 AD-76180 132 13.9 124.2 16.8 1109-1129 AD-7618167.1 5.9 99.1 0.3 1541-1561 AD-76182 110.4 17.9 102 5.1 824-844 AD-7618369.8 9.9 100.3 6.9 893-913 AD-76184 62 6.3 99.4 7.1 965-985 AD-76185119.5 33.1 92.8 4.4 530-550 AD-76186 52.4 4.6 94.7 2.7 1617-1637AD-76187 116.7 6.4 117.7 9.4 1633-1653 AD-76188 60.4 5.8 106.2 4.81185-1205 AD-76189 89.9 2.1 102.9 8.4 1473-1493 AD-76190 83.5 3.5 104.56.5 1545-1565 AD-76191 80.9 3.3 96.4 7.2 1110-1130 AD-76192 93.9 3.9 1035.4 1472-1492 AD-76193 99.3 3.5 95.1 10.2 1610-1630 AD-76194 192.1 4.9118 12.1 1005-1025 AD-76195 86.1 3.3 106.7 8.4 1342-1362 AD-76196 18423.4 114.8 6.7 1006-1026 AD-76197 55.3 4.4 111.4 14.2 1530-1550 AD-7619866.7 3.6 97.1 13.1 1351-1371 AD-76199 54.5 2 91.8 4.2 358-378 AD-7620063.9 10.1 88 10.6 1174-1194 AD-76201 150.6 4.6 113.6 18.3 559-579AD-76202 64.7 1.4 95.7 13.7 820-840 AD-76203 41.3 1.7 93 2.1 238-258AD-76204 67.5 6.9 101.3 3.6 549-569 AD-76205 73.1 9.5 87.6 7.8 1176-1196AD-76206 86.4 3.7 107.7 17 1036-1056 AD-76207 131.4 22.4 99.7 10.5742-762 AD-76208 46.5 12 103 13.2 209-229 AD-76209 43.8 5.2 98.1 13.9254-274 AD-76210 41.6 7.3 94.4 6 1066-1086 AD-76211 152.3 9.4 125.1 3.41396-1416 AD-76212 59.2 12.7 82.3 23.4 604-624 AD-76213 71.1 4.9 94.911.6 440-460 AD-76214 109.8 5.9 102.8 8.4 1096-1116 AD-76215 70.2 7.5103.2 25.4 1961-1981 AD-76216 62.8 9.5 107.7 15.1 882-902 AD-76217 68.81.7 94.5 2.4 1453-1473 Mock 117.3 16.3 106.9 9.5

TABLE 14 Modified Sense and Antisense Strand Sequences of IGFALS dsRNAsSense SEQ Antisense SEQ SEQ Duplex Oligo ID Oligo ID ID Name NameSense Oligo Sequence NO Name Antisense Oligo Sequence NOmRNA target sequence NO AD-76171 A-152158 csasauuaAfaGfGfCfaaaggcaauaL961531 A-152159 VPusAfsuugCfcUfUfugccUfuUfaauugsasu 1578AUCAAUUAAAGGCAAAGGCAAUC 1625 AD-76172 A-152160ascsaccuAfcAfAfCfaacaucaccaL96 1532 A-152161VPusGfsgugAfuGfUfuguuGfuAfggugusasc 1579 GUACACCUACAACAACAUCACCU 1626AD-76173 A-152162 usascaccUfaCfAfAfcaacaucacaL96 1533 A-152163VPusGfsugaUfgUfUfguugUfaGfguguascsg 1580 CGUACACCUACAACAACAUCACC 1627AD-76174 A-152164 csasucaaGfgCfAfAfacguguucgaL96 1534 A-152165VPusCfsgaaCfaCfGfuuugCfcUfugaugsgsc 1581 GCCAUCAAGGCAAACGUGUUCGU 1628AD-76175 A-152166 csasccuaCfaAfCfAfacaucaccuaL96 1535 A-152167VPusAfsgguGfaUfGfuuguUfgUfaggugsusa 1582 UACACCUACAACAACAUCACCUG 1629AD-76176 A-152168 csgsuacaCfcUfAfCfaacaacaucaL96 1536 A-152169VPusGfsaugUfuGfUfuguaGfgUfguacgscsg 1583 CGCGUACACCUACAACAACAUCA 1630AD-76177 A-152170 gsusacacCfuAfCfAfacaacaucaaL96 1537 A-152171VPusUfsgauGfuUfGfuuguAfgGfuguacsgsc 1584 GCGUACACCUACAACAACAUCAC 1631AD-76178 A-152172 gsascucuUfcCfUfCfaaggacaacaL96 1538 A-152173VPusGfsuugUfcCfUfugagGfaAfgagucsgsg 1585 CCGACUCUUCCUCAAGGACAACG 1632AD-76179 A-152174 asgsccuuCfcAfGfAfaccucuccaaL96 1539 A-152175VPusUfsggaGfaGfGfuucuGfgAfaggcusgsc 1586 GCAGCCUUCCAGAACCUCUCCAG 1633AD-76180 A-152176 uscsacgcUfaGfAfCfcacaaccagaL96 1540 A-152177VPusCfsuggUfuGfUfggucUfaGfcgugasgsc 1587 GCUCACGCUAGACCACAACCAGC 1634AD-76181 A-152178 usgsgacgUfcUfCfGfcacaaccgcaL96 1541 A-152179VPusGfscggUfuGfUfgcgaGfaCfguccasgsc 1588 GCUGGACGUCUCGCACAACCGCC 1635AD-76182 A-152180 ascscuggAfcCfGfCfaaccucaucaL96 1542 A-152181VPusGfsaugAfgGfUfugcgGfuCfcaggusasg 1589 CUACCUGGACCGCAACCUCAUCG 1636AD-76183 A-152182 usgsgaccUfgUfCfCfcacaaccgcaL96 1543 A-152183VPusGfscggUfuGfUfgggaCfaGfguccasgsc 1590 GCUGGACCUGUCCCACAACCGCG 1637AD-76184 A-152184 usgscggcUfgUfCfCfcacaacgccaL96 1544 A-152185VPusGfsgcgUfuGfUfgggaCfaGfccgcasgsc 1591 GCUGCGGCUGUCCCACAACGCCA 1638AD-76185 A-152186 csgscucgGfcCfUfCfagcaacaacaL96 1545 A-152187VPusGfsuugUfuGfCfugagGfcCfgagcgsasg 1592 CUCGCUCGGCCUCAGCAACAACC 1639AD-76186 A-152188 cscsucagGfaAfCfAfacucacugcaL96 1546 A-152189VPusGfscagUfgAfGfuuguUfcCfugaggscsu 1593 AGCCUCAGGAACAACUCACUGCG 1640AD-76187 A-152190 csusgeggAfcCfUfUfcacgccgcaaL96 1547 A-152191VPusUfsgegGfcGfUfgaagGfuCfcgcagsusg 1594 CACUGCGGACCUUCACGCCGCAG 1641AD-76188 A-152192 csesucucUfgGfGfAfacugucuccaL96 1548 A-152193VPusGfsgagAfcAfGfuuccCfaGfagaggsusu 1595 AACCUCUCUGGGAACUGUCUCCG 1642AD-76189 A-152194 gscsucucCfcGfCfAfaccgccuggaL96 1549 A-152195VPusCfscagGfcGfGfuugcGfgGfagagcsasg 1596 CUGCUCUCCCGCAACCGCCUGGC 1643AD-76190 A-152196 csgsucucGfcAfCfAfaccgccuggaL96 1550 A-152197VPusCfscagGfcGfGfuuguGfcGfagacgsusc 1597 GACGUCUCGCACAACCGCCUGGA 1644AD-76191 A-152198 csascgcuAfgAfCfCfacaaccagcaL96 1551 A-152199VPusGfscugGfuUfGfugguCfuAfgcgugsasg 1598 CUCACGCUAGACCACAACCAGCU 1645AD-76192 A-152200 usgscucuCfcCfGfCfaaccgccugaL96 1552 A-152201VPusCfsaggCfgGfUfugegGfgAfgagcasgsc 1599 GCUGCUCUCCCGCAACCGCCUGG 1646AD-76193 A-152202 ascscucaGfcCfUfCfaggaacaacaL96 1553 A-152203VPusGfsuugUfuCfCfugagGfcUfgaggusasg 1600 CUACCUCAGCCUCAGGAACAACU 1647AD-76194 A-152204 csasccuuCfaAfGfGfaccugcacuaL96 1554 A-152205VPusAfsgugCfaGfGfuccuUfgAfaggugscsg 1601 CGCACCUUCAAGGACCUGCACUU 1648AD-76195 A-152206 gsgsccucGfuGfGfGfcauugaggaaL96 1555 A-152207VPusUfsccuCfaAfUfgcccAfcGfaggccsgsu 1602 ACGGCCUCGUGGGCAUUGAGGAG 1649AD-76196 A-152208 ascscuucAfaGfGfAfccugcacuuaL96 1556 A-152209VPusAfsaguGfcAfGfguccUfuGfaaggusgsc 1603 GCACCUUCAAGGACCUGCACUUC 1650AD-76197 A-152210 gsgsccuuCfuGfGfCfuggacgucuaL96 1557 A-152211VPusAfsgacGfuCfCfagccAfgAfaggccscsg 1604 CGGGCCUUCUGGCUGGACGUCUC 1651AD-76198 A-152212 gsgscauuGfaGfGfAfgcagagccuaL96 1558 A-152213VPusAfsggcUfcUfGfcuccUfcAfaugccscsa 1605 UGGGCAUUGAGGAGCAGAGCCUG 1652AD-76199 A-152214 gscscuucCfaGfAfAfccucuccagaL96 1559 A-152215VPusCfsuggAfgAfGfguucUfgGfaaggcsusg 1606 CAGCCUUCCAGAACCUCUCCAGC 1653AD-76200 A-152216 gscsggucAfuGfAfAfccucucuggaL96 1560 A-152217VPusCfscagAfgAfGfguucAfuGfaccgcscsa 1607 UGGCGGUCAUGAACCUCUCUGGG 1654AD-76201 A-152218 asgsgcugGfaGfGfAfcgggcucuuaL96 1561 A-152219VPusAfsagaGfcCfCfguccUfcCfagccusgsc 1608 GCAGGCUGGAGGACGGGCUCUUC 1655AD-76202 A-152220 csuscuacCfuGfGfAfccgcaaccuaL96 1562 A-152221VPusAfsgguUfgCfGfguccAfgGfuagagsusu 1609 AACUCUACCUGGACCGCAACCUC 1656AD-76203 A-152222 gscsggauGfaGfCfUfcagcgucuuaL96 1563 A-152223VPusAfsagaCfgCfUfgagcUfcAfuccgcsgsu 1610 ACGCGGAUGAGCUCAGCGUCUUC 1657AD-76204 A-152224 cscsgucuGfaGfCfAfggcuggaggaL96 1564 A-152225VPusCfscucCfaGfCfcugcUfcAfgacggsusu 1611 AACCGUCUGAGCAGGCUGGAGGA 1658AD-76205 A-152226 gsgsucauGfaAfCfCfucucugggaaL96 1565 A-152227VPusUfscccAfgAfGfagguUfcAfugaccsgsc 1612 GCGGUCAUGAACCUCUCUGGGAA 1659AD-76206 A-152228 csusgcagCfuGfGfGfccacaaccgaL96 1566 A-152229VPusCfsgguUfgUfGfgcccAfgCfugcagscsu 1613 AGCUGCAGCUGGGCCACAACCGC 1660AD-76207 A-152230 csgsggagCfuGfGfAfccugagcagaL96 1567 A-152231VPusCfsugcUfcAfGfguccAfgCfucccgsgsa 1614 UCCGGGAGCUGGACCUGAGCAGG 1661AD-76208 A-152232 cscsgccuGfuGfUfCfugcagcuacaL96 1568 A-152233VPusGfsuagCfuGfCfagacAfcAfggcggscsc 1615 GGCCGCCUGUGUCUGCAGCUACG 1662AD-76209 A-152234 uscsuucuGfcAfGfCfuccaggaacaL96 1569 A-152235VPusGfsuucCfuGfGfagcuGfcAfgaagascsg 1616 CGUCUUCUGCAGCUCCAGGAACC 1663AD-76210 A-152236 csusggcuGfaGfCfGfcagcuuugaaL96 1570 A-152237VPusUfscaaAfgCfUfgcgcUfcAfgccagscsu 1617 AGCUGGCUGAGCGCAGCUUUGAG 1664AD-76211 A-152238 csuscgacCfuGfAfCfcuccaaccaaL96 1571 A-152239VPusUfsgguUfgGfAfggucAfgGfucgagscsu 1618 AGCUCGACCUGACCUCCAACCAG 1665AD-76212 A-152240 csuscaacCfuCfGfGfcuggaauagaL96 1572 A-152241VPusCfsuauUfcCfAfgccgAfgGfuugagsgsu 1619 ACCUCAACCUCGGCUGGAAUAGC 1666AD-76213 A-152242 gscscuagAfgAfAfCfcugugccacaL96 1573 A-152243VPusGfsuggCfaCfAfgguuCfuCfuaggcscsc 1620 GGGCCUAGAGAACCUGUGCCACC 1667AD-76214 A-152244 csasgcuuGfaGfGfUfgcucacgcuaL96 1574 A-152245VPusAfsgcgUfgAfGfcaccUfcAfagcugscsc 1621 GGCAGCUUGAGGUGCUCACGCUA 1668AD-76215 A-152246 csasggucCfuCfAfGfuguccucagaL96 1575 A-152247VPusCfsugaGfgAfCfacugAfgGfaccugsusc 1622 GACAGGUCCUCAGUGUCCUCAGG 1669AD-76216 A-152248 gscsugcgAfuGfGfCfuggaccuguaL96 1576 A-152249VPusAfscagGfuCfCfagccAfuCfgcagcsgsc 1623 GCGCUGCGAUGGCUGGACCUGUC 1670AD-76217 A-152250 gsgscaagCfuGfGfAfguaccugcuaL96 1577 A-152251VPusAfsgcaGfgUfAfcuccAfgCfuugccscsa 1624 UGGGCAAGCUGGAGUACCUGCUG 1671

Example 10-IGF-1 Transcripts, siRNA Design, and siRNA Screening

Bioinformatics

A set of siRNAs targeting the human IGF1, (“insulin like growth factor1”, NCBI refseqID: NM_000618; NCBI GeneID: 3479 (SEQ ID NO:13), as wellas toxicology-species IGF1 orthologs (cynomolgus monkey: XM_005572039)were designed using custom R and Python scripts. The human NM_000618REFSEQ mRNA, version 3, has a length 7321 of bases.

The rationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 10through the end was determined with a linear model derived the directmeasure of mRNA knockdown from more than 20,000 distinct siRNA designstargeting a large number of vertebrate genes. Subsets of the IGF1 siRNAswere designed with perfect or near-perfect matches between human andcynomolgus monkey. For each strand of the siRNA, a custom Python scriptwas used in a brute force search to measure the number and positions ofmismatches between the siRNA and all potential alignments in the targetspecies transcriptome. Extra weight was given to mismatches in the seedregion, defined here as positions 2-9 of the antisense oligonucleotide,as well the cleavage site of the siRNA, defined here as positions 10-11of the antisense oligonucleotide. The relative weight of the mismatcheswas 2.8; 1.2:1 for seed mismatches, cleavage site, and other positionsup through antisense position 19. Mismatches in the first position wereignored. A specificity score was calculated for each strand by summingthe value of each weighted mismatch. Preference was given to siRNAswhose antisense score in human was >=2.2 and predicted efficacywas >=50% knockdown of the transcript.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Three humanIGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2vector. Construct one contained sequence based on NM_001111285 (SEQ IDNO:1672), while constructs two and three contained sequence based onNM_000618 (SEQ ID NOs: 1673 and 1674). Dual-luciferase plasmids wereco-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax(Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384well plate, 0.1 μl of Lipofectamine was added to 5 ng of plasmid vectorand siRNA in 15 μl of Opti-MEM and allowed to complex at roomtemperature for 15 minutes. The mixture was then added to the cellsresuspended in 35 ul of fresh complete media. Cells were incubated for48 hours before luciferase was measured. Screen was performed at 10 nMand 0.1 nM final duplex concentration.

Dual-Glo® Luciferase Assay

48 hours after the siRNAs were transfected, Firefly (transfectioncontrol) and Renilla (fused to IGF1 target sequence in 3′ UTR)luciferase were measured. First, media was removed from cells. ThenFirefly luciferase activity was measured by adding 20 ul of Dual-Glo®Luciferase Reagent mixed with 20 ul of complete media to each well. Themixture was incubated at room temperature for 30 minutes beforeluminescense (500 nm) was measured on a Spectramax (Molecular Devices)to detect the Firefly luciferase signal. Renilla luciferase activity wasmeasured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo®Reagent to each well and the plates were incubated for 20 minutes beforeluminescence was again measured to determine the Renilla luciferasesignal. The Dual-Glo® Stop & Glo® Reagent quenched the fireflyluciferase signal and sustained luminescence for the Renilla luciferasereaction. siRNA activity was determined by normalizing the Renilla(IGF1) signal to the Firefly (control) signal within each well. Themagnitude of siRNA activity was then assessed relative to cells thatwere transfected with the same vector but were not treated with siRNA orwere treated with a non-targeting siRNA. All transfections were done inquadruplicates.

TABLE 15 Unmodified Sense and Antisense Strand Sequences of IGF-1dsRNAsSense Position SEQ Antisense Position SEQ Duplex Oligo in NM_ ID Oligoin NM_ ID Name Name Sense Sequence 000618.3 NO Name Antisense Sequence000618.3 NO AD-75740 A-151647 AAUGUAACUAAUUGAAUCAUA 7181-7201 1675A-151648 UAUGAUUCAAUUAGUUACAUUUU 7179-7201 1768 AD-75741 A-151649UAAGAAAGUACUUGACUAAAA 4362-4382 1676 A-151650 UUUUAGUCAAGUACUUUCUUAAA4360-4382 1769 AD-75747 A-151661 CUUUAUAAUUCUUGAAUGAGA 3765-3785 1677A-151662 UCUCAUUCAAGAAUUAUAAAGCA 3763-3785 1770 AD-75748 A-151663AGAUAGAAAUGUAUGUUUGAA 5223-5243 1678 A-151664 UUCAAACAUACAUUUCUAUCUAG5221-5243 1771 AD-75749 A-151665 UUCCACAAUCCUUAGAAUCUA 6512-6532 1679A-151666 UAGAUUCUAAGGAUUGUGGAAGG 6510-6532 1772 AD-75750 A-151667UAUCAAAACCUUUCAAAUAUA 6831-6851 1680 A-151668 UAUAUUUGAAAGGUUUUGAUAUU6829-6851 1773 AD-75751 A-151669 ACAAGUAAACAUUCCAACAUA  824-844 1681A-151670 UAUGUUGGAAUGUUUACUUGUGU  822-844 1774 AD-75755 A-151677ACAUAGAAAGUUUCUUCAACA 1410-1430 1682 A-151678 UGUUGAAGAAACUUUCUAUGUUU1408-1430 1775 AD-75757 A-151681 CAGUCAACAAGUAUUUUAACA 3122-3142 1683A-151682 UGUUAAAAUACUUGUUGACUGAA 3120-3142 1776 AD-75759 A-151685CUCAAGCUGUCUACUUACAUA 6769-6789 1684 A-151686 UAUGUAAGUAGACAGCUUGAGGU6767-6789 1777 AD-75760 A-151687 GAAUUGUUUCCUUAUUUGCAA 1085-1105 1685A-151688 UUGCAAAUAAGGAAACAAUUCAU 1083-1105 1778 AD-75761 A-151689AUCUGUCUUAGUUGAAAAGCA 7071-7091 1686 A-151690 UGCUUUUCAACUAAGACAGAUGU7069-7091 1779 AD-75765 A-151697 AAUUAGCAAUAUAUUAUCCAA 4632-4652 1687A-151698 UUGGAUAAUAUAUUGCUAAUUUU 4630-4652 1780 AD-75766 A-151699AAUUGAAUCAUUAUCUUACAA 7190-7210 1688 A-151700 UUGUAAGAUAAUGAUUCAAUUAG7188-7210 1781 AD-75769 A-151705 UUUAUCAAUAAUGUUCUAUAA  908-928 1689A-151706 UUAUAGAACAUUAUUGAUAAAAG  906-928 1782 AD-75772 A-151711GUAAAAGAAACUAUACAUCAA 3213-3233 1690 A-151712 UUGAUGUAUAGUUUCUUUUACAU3211-3233 1783 AD-75774 A-151715 AAUGAGGAAUAAUAAGUUAAA 5173-5193 1691A-151716 UUUAACUUAUUAUUCCUCAUUCU 5171-5193 1784 AD-75776 A-151719CUCUGUGAAUGUGUUUUAUCA 2737-2757 1692 A-151720 UGAUAAAACACAUUCACAGAGAG2735-2757 1785 AD-75778 A-151723 GUUCCUUCAAAUGAUGAGUUA 1438-1458 1693A-151724 UAACUCAUCAUUUGAAGGAACUC 1436-1458 1786 AD-75779 A-151725CAGGAUAAAGAUAUCAAUUUA 5722-5742 1694 A-151726 UAAAUUGAUAUCUUUAUCCUGUA5720-5742 1787 AD-75787 A-151741 CAUGUCCUCCUCGCAUCUCUA  297-317 1695A-151742 UAGAGAUGCGAGGAGGACAUGGU  295-317 1788 AD-75787 A-151741CAUGUCCUCCUCGCAUCUCUA  297-317 1696 A-151742 UAGAGAUGCGAGGAGGACAUGGU 295-317 1789 AD-75788 A-151743 UUCAACAAGCCCACAGGGUAA  436-456 1697A-151744 UUACCCUGUGGGCUUGUUGAAAU  434-456 1790 AD-75788 A-151743UUCAACAAGCCCACAGGGUAA  436-456 1698 A-151744 UUACCCUGUGGGCUUGUUGAAAU 434-456 1791 AD-75789 A-151745 UCCUCGCAUCUCUUCUACCUA  304-324 1699A-151746 UAGGUAGAAGAGAUGCGAGGAGG  302-324 1792 AD-75789 A-151745UCCUCGCAUCUCUUCUACCUA  304-324 1700 A-151746 UAGGUAGAAGAGAUGCGAGGAGG 302-324 1793 AD-75790 A-151747 GGGGCUUUUAUUUCAACAAGA  425-445 1701A-151748 UCUUGUUGAAAUAAAAGCCCCUG  423-445 1794 AD-75790 A-151747GGGGCUUUUAUUUCAACAAGA  425-445 1702 A-151748 UCUUGUUGAAAUAAAAGCCCCUG 423-445 1795 AD-75791 A-151749 GAUGCUCUUCAGUUCGUGUGA  397-417 1703A-151750 UCACACGAACUGAAGAGCAUCCA  395-417 1796 AD-75791 A-151749GAUGCUCUUCAGUUCGUGUGA  397-417 1704 A-151750 UCACACGAACUGAAGAGCAUCCA 395-417 1797 AD-75792 A-151751 UGGAUGCUCUUCAGUUCGUGA  395-415 1705A-151752 UCACGAACUGAAGAGCAUCCACC  393-415 1798 AD-75792 A-151751UGGAUGCUCUUCAGUUCGUGA  395-415 1706 A-151752 UCACGAACUGAAGAGCAUCCACC 393-415 1799 AD-75793 A-151753 GGUGGAUGCUCUUCAGUUCGA  393-413 1707A-151754 UCGAACUGAAGAGCAUCCACCAG  391-413 1800 AD-75793 A-151753GGUGGAUGCUCUUCAGUUCGA  393-413 1708 A-151754 UCGAACUGAAGAGCAUCCACCAG 391-413 1801 AD-75794 A-151755 GCUGGUGGAUGCUCUUCAGUA  390-410 1709A-151756 UACUGAAGAGCAUCCACCAGCUC  388-410 1802 AD-75794 A-151755GCUGGUGGAUGCUCUUCAGUA  390-410 1710 A-151756 UACUGAAGAGCAUCCACCAGCUC 388-410 1803 AD-75795 A-151757 CUGGUGGAUGCUCUUCAGUUA  391-411 1711A-151758 UAACUGAAGAGCAUCCACCAGCU  389-411 1804 AD-75795 A-151757CUGGUGGAUGCUCUUCAGUUA  391-411 1712 A-151758 UAACUGAAGAGCAUCCACCAGCU 389-411 1805 AD-77120 A-154752 CCAAAAUGCACUGAUGUAAAA 6586-6606 1713A-154753 UUUUACAUCAGUGCAUUUUGGGC 6584-6606 1806 AD-77121 A-154754UCCAGUUGCACUAAAUUCCUA 1009-1029 1714 A-154755 UAGGAAUUUAGUGCAACUGGAUC1007-1029 1807 AD-77122 A-154756 CAUUCUUCAACUAUCUUUGAA 6325-6345 1715A-154757 UUCAAAGAUAGUUGAAGAAUGAG 6323-6345 1808 AD-77123 A-154758ACAUUCCAACAUUGUCUUUAA  832-852 1716 A-154759 UUAAAGACAAUGUUGGAAUGUUU 830-852 1809 AD-77124 A-154760 GGCUUAGAAUAAAAGAUGUAA 4280-4300 1717A-154761 UUACAUCUUUUAUUCUAAGCCUU 4278-4300 1810 AD-77125 A-154762UUUCAAGAUAUUUGUAAAAGA 3789-3809 1718 A-154763 UCUUUUACAAAUAUCUUGAAAUU3787-3809 1811 AD-77126 A-154764 AUAAGCAUAUUUUGAAAAUGA 7221-7241 1719A-154765 UCAUUUUCAAAAUAUGCUUAUUA 7219-7241 1812 AD-77127 A-154766GUUUUCAAUGCUAGUGUUUAA 5553-5573 1720 A-154767 UUAAACACUAGCAUUGAAAACAA5551-5573 1813 AD-77128 A-154768 CAAUGAAAUACACAAGUAAAA  813-833 1721A-154769 UUUUACUUGUGUAUUUCAUUGGG  811-833 1814 AD-77129 A-154770CAAAAGUCCACUGAUGCAAAA 1764-1784 1722 A-154771 UUUUGCAUCAGUGGACUUUUGUG1762-1784 1815 AD-77130 A-154772 AACAUAGAAAGUUUCUUCAAA 1409-1429 1723A-154773 UUUGAAGAAACUUUCUAUGUUUA 1407-1429 1816 AD-77131 A-154774AACCUAAUUAGUAACUUUCCA 1465-1485 1724 A-154775 UGGAAAGUUACUAAUUAGGUUGC1463-1485 1817 AD-77132 A-154776 GACUUAUUUCCUUCACUUAAA 3442-3462 1725A-154777 UUUAAGUGAAGGAAAUAAGUCAU 3440-3462 1818 AD-77133 A-154778GGCUCUCAAACUUAGCAAAAA 4614-4634 1726 A-154779 UUUUUGCUAAGUUUGAGAGCCAA4612-4634 1819 AD-77134 A-154780 ACAAAGAUAAGUGAAAAGAGA 2312-2332 1727A-154781 UCUCUUUUCACUUAUCUUUGUAU 2310-2332 1820 AD-77135 A-154782GUAGUGUACUGUUCACCAAAA 4525-4545 1728 A-154783 UUUUGGUGAACAGUACACUACUU4523-4545 1821 AD-77136 A-154784 UCACCAACUCAUAGCAAAGUA 6663-6683 1729A-154785 UACUUUGCUAUGAGUUGGUGAGU 6661-6683 1822 AD-77137 A-154786AAAAUAACUCAUUAUACCAAA 3577-3597 1730 A-154787 UUUGGUAUAAUGAGUUAUUUUCA3575-3597 1823 AD-77138 A-154788 AAGAAAGAAUCUUUCCAUUCA 5344-5364 1731A-154789 UGAAUGGAAAGAUUCUUUCUUCU 5342-5364 1824 AD-77139 A-154790AUUUCAAGAUAUUUGUAAAAA 3788-3808 1732 A-154791 UUUUUACAAAUAUCUUGAAAUUG3786-3808 1825 AD-77140 A-154792 AGCUGAAACUCUAGAAUUAAA 5908-5928 1733A-154793 UUUAAUUCUAGAGUUUCAGCUUG 5906-5928 1826 AD-77141 A-154794AAAGAUAAAUCUGAUUUAUGA 5394-5414 1734 A-154795 UCAUAAAUCAGAUUUAUCUUUUG5392-5414 1827 AD-77142 A-154796 UAUUAAAUUAAUUCUGAUUGA 7099-7119 1735A-154797 UCAAUCAGAAUUAAUUUAAUAAA 7097-7119 1828 AD-77143 A-154798AAGAAUGAGCUUUCAACUCAA 3825-3845 1736 A-154799 UUGAGUUGAAAGCUCAUUCUUAC3823-3845 1829 AD-77144 A-154800 GUUUGAUAACAUUUAAAAGAA  780-800 1737A-154801 UUCUUUUAAAUGUUAUCAAACUU  778-800 1830 AD-77145 A-154802UGUUUUAAACAUAGAAAGUUA 1402-1422 1738 A-154803 UAACUUUCUAUGUUUAAAACAUA1400-1422 1831 AD-77146 A-154804 UAAAUAACCCAUUCAUAGCAA 2110-2130 1739A-154805 UUGCUAUGAAUGGGUUAUUUAUA 2108-2130 1832 AD-77147 A-154806CACAUAGACUCUUUAAAACUA 5196-5216 1740 A-154807 UAGUUUUAAAGAGUCUAUGUGGG5194-5216 1833 AD-77148 A-154808 AAGUCACAAAGUUAUCUUCUA 2568-2588 1741A-154809 UAGAAGAUAACUUUGUGACUUUU 2566-2588 1834 AD-77149 A-154810UAAAGAAAGAAUUUAUGAGAA 5011-5031 1742 A-154811 UUCUCAUAAAUUCUUUCUUUAUC5009-5031 1835 AD-77150 A-154812 GAAAUGUAUGUUUGACUUGUA 5228-5248 1743A-154813 UACAAGUCAAACAUACAUUUCUA 5226-5248 1836 AD-77151 A-154814UUUAUAAGACCUUCCUGUUAA 5611-5631 1744 A-154815 UUAACAGGAAGGUCUUAUAAAAU5609-5631 1837 AD-77152 A-154816 CUGUGAAUGUGUUUUAUCCAA 2739-2759 1745A-154817 UUGGAUAAAACACAUUCACAGAG 2737-2759 1838 AD-77153 A-154818UUAAUUCUGAUUGUAUUUGAA 7106-7126 1746 A-154819 UUCAAAUACAAUCAGAAUUAAUU7104-7126 1839 AD-77154 A-154820 UGUCAUCUACCUACCUCAAAA 1982-2002 1747A-154821 UUUUGAGGUAGGUAGAUGACAUA 1980-2002 1840 AD-77155 A-154822CGUGAAAGCAAAACAAUAGGA 1634-1654 1748 A-154823 UCCUAUUGUUUUGCUUUCACGUA1632-1654 1841 AD-77156 A-154824 AACAACAAUUCUAUAGAUAGA 4438-4458 1749A-154825 UCUAUCUAUAGAAUUGUUGUUUU 4436-4458 1842 AD-77157 A-154826UAAAGUAUUAUUUGAACUUUA 3920-3940 1750 A-154827 UAAAGUUCAAAUAAUACUUUAAU3918-3940 1843 AD-77158 A-154828 AGAAAGAAUUUAUGAGAAAUA 5014-5034 1751A-154829 UAUUUCUCAUAAAUUCUUUCUUU 5012-5034 1844 AD-77159 A-154830AAAUGCACUGAUGUAAAGUAA 6589-6609 1752 A-154831 UUACUUUACAUCAGUGCAUUUUG6587-6609 1845 AD-77160 A-154832 AUUAAUUCUGAUUGUAUUUGA 7105-7125 1753A-154833 UCAAAUACAAUCAGAAUUAAUUU 7103-7125 1846 AD-77161 A-154834CAAUAAUGUUCUAUAGAAAAA  913-933 1754 A-154835 UUUUUCUAUAGAACAUUAUUGAU 911-933 1847 AD-77162 A-154836 GAUUUUCAAUUUGAUUUUGAA 2269-2289 1755A-154837 UUCAAAAUCAAAUUGAAAAUCAA 2267-2289 1848 AD-77163 A-154840UUGAUUUUGAAUUCUGCAUUA 2279-2299 1756 A-154841 UAAUGCAGAAUUCAAAAUCAAAU2277-2299 1849 AD-77164 A-154842 UUUCAAUUUGAUUUUGAAUUA 2272-2292 1757A-154843 UAAUUCAAAAUCAAAUUGAAAAU 2270-2292 1850 AD-77165 A-154844GUGUCUGUGUAUCAUGAAAAA 6798-6818 1758 A-154845 UUUUUCAUGAUACACAGACACAG6796-6818 1851 AD-77166 A-154846 GGUUUUAUGAAUACAAAGAUA 2300-2320 1759A-154847 UAUCUUUGUAUUCAUAAAACCAA 2298-2320 1852 AD-77167 A-154848GCAGAUCAAGAUUUUCUCAUA 2837-2857 1760 A-154849 UAUGAGAAAAUCUUGAUCUGCAG2835-2857 1853 AD-77168 A-154850 AACUAAUUGAAUCAUUAUCUA 7186-7206 1761A-154851 UAGAUAAUGAUUCAAUUAGUUAC 7184-7206 1854 AD-77169 A-154852UGGUUUAUGAAUUGUUUCCUA 1077-1097 1762 A-154853 UAGGAAACAAUUCAUAAACCACU1075-1097 1855 AD-77170 A-154854 UAGUAUAAUGGUGCUAUUUUA 1574-1594 1763A-154855 UAAAAUAGCACCAUUAUACUAAA 1572-1594 1856 AD-77171 A-154856CUGUUUAAUAAGCAUAUUUUA 7214-7234 1764 A-154857 UAAAAUAUGCUUAUUAAACAGUA7212-7234 1857 AD-77172 A-154858 AAAUAUCAAAACCUUUCAAAA 6828-6848 1765A-154859 UUUUGAAAGGUUUUGAUAUUUUG 6826-6848 1858 AD-77173 A-154860ACAGAUGUAAAAGAAACUAUA 3207-3227 1766 A-154861 UAUAGUUUCUUUUACAUCUGUCC3205-3227 1859 AD-77174 A-154862 AACUUUGAGGCCAAUCAUUUA 1373-1393 1767A-154863 UAAAUGAUUGGCCUCAAAGUUGC 1371-1393 1860

TABLE 16 IGF-1 in vitro 10 nM and 0.1 nM screen Duplex 10 nM 10 nM 0.1nM 0.1 nM Position in Name AVG STD AVG STD NM_000618.3 AD-75740 2.9 1.120.9 1.6 7179-7201 AD-75741 9.2 1.5 55.6 9.3 4360-4382 AD-75747 50.3 6.867.7 8.8 3763-3785 AD-75748 8.4 1.1 36 10.1 5221-5243 AD-75749 7.8 1.365.9 3 6510-6532 AD-75750 4.4 1.3 48 9.3 6829-6851 AD-75751 5.7 1.4 51.79.4 822-844 AD-75755 29.4 7.2 60.9 3.8 1408-1430 AD-75757 32.4 5.7 62.37.9 3120-3142 AD-75759 9.8 4.3 86.5 11.1 6767-6789 AD-75760 32.2 7.856.2 7.1 1083-1105 AD-75761 3.5 1.3 53.3 4.3 7069-7091 AD-75765 7.6 1.956.5 8.6 4630-4652 AD-75766 3.3 2.4 35.4 7.4 7188-7210 AD-75769 12.4 3.133.6 3.9 906-928 AD-75772 36.1 5.2 79.9 19.3 3211-3233 AD-75774 14 1.251.8 7.8 5171-5193 AD-75776 41.1 11.6 84.2 10.3 2735-2757 AD-75778 42.37.4 56.4 6.6 1436-1458 AD-75779 8.7 1.6 53.7 9.1 5720-5742 AD-75787 59.55.5 96.5 6.2 295-317 AD-75787 57.5 12.9 99.5 10.4 295-317 AD-75788 38.55.2 82.2 9.3 434-456 AD-75788 28.1 2.1 88.3 4.4 434-456 AD-75789 58 11.581.6 3 302-324 AD-75789 65.1 16.2 93.6 5.6 302-324 AD-75790 54.7 5.990.1 1.7 423-445 AD-75790 64.1 6.1 92.6 4.3 423-445 AD-75791 17.4 5 78.83.9 395-417 AD-75791 19.5 2.1 87.5 7.1 395-417 AD-75792 12.7 1.5 63.76.2 393-415 AD-75792 13.9 2 77.8 12.9 393-415 AD-75793 23.6 4.1 78.5 4.7391-413 AD-75793 27.8 4.1 90.9 11.6 391-413 AD-75794 40.6 3.1 89 12.3388-410 AD-75794 46.7 4.4 91.1 9.6 388-410 AD-75795 32.2 8.2 75.2 6.3389-411 AD-75795 27.3 5.3 77.7 7.3 389-411 AD-77120 17.7 1.8 83.5 3.76584-6606 AD-77121 28.3 3.4 69 5.9 1007-1029 AD-77122 19.3 6.4 80.4 10.56323-6345 AD-77123 21.2 4.1 46.9 8 830-852 AD-77124 13.9 1.5 35.3 5.84278-4300 AD-77125 44.5 10.3 52.4 6.9 3787-3809 AD-77126 4.5 3 23.6 3.97219-7241 AD-77127 8.3 1.5 43.1 4.9 5551-5573 AD-77128 13.6 4.1 46.6 5.3811-833 AD-77129 68.5 7.6 99.9 8.2 1762-1784 AD-77130 32.1 5.8 41.4 3.41407-1429 AD-77131 36 8.5 53.8 7.2 1463-1485 AD-77132 44.3 8.2 71.7 5.13440-3462 AD-77133 21.4 6.4 88.9 5.8 4612-4634 AD-77134 37.9 2.4 82.48.7 2310-2332 AD-77135 19.6 5 91.8 17 4523-4545 AD-77136 32.3 8 89.5 3.56661-6683 AD-77137 36.8 3.3 67.7 7.7 3575-3597 AD-77138 2.6 1.9 71.3 6.35342-5364 AD-77139 45.6 2.5 70.4 8.2 3786-3808 AD-77140 17.1 3.2 65.46.1 5906-5928 AD-77141 56.3 17.6 112.7 8.2 5392-5414 AD-77142 18.1 3.765.7 8.4 7097-7119 AD-77143 45.7 10.1 93.6 11 3823-3845 AD-77144 5.5 243.5 7.6 778-800 AD-77145 59.6 11.1 43.8 5.1 1400-1422 AD-77146 27.7 5.864.8 7.7 2108-2130 AD-77147 6.6 5.4 75 10.2 5194-5216 AD-77148 37.6 6.765.9 8 2566-2588 AD-77149 5.5 0.7 35.8 5.2 5009-5031 AD-77150 1.1 2.348.2 5.1 5226-5248 AD-77151 9.1 2.3 69.2 5.9 5609-5631 AD-77152 52.6 5.993.7 6.5 2737-2759 AD-77153 10.4 1 37.8 6.1 7104-7126 AD-77154 57.5 14.5101.7 11.5 1980-2002 AD-77155 63.3 8.5 53 3.9 1632-1654 AD-77156 14.8 347.9 8.9 4436-4458 AD-77158 1.3 1 44.3 0.9 5012-5034 AD-77159 20.5 4.992.3 17.2 6587-6609 AD-77160 10.8 5.2 39.3 4.9 7103-7125 AD-77161 26.11.8 47.3 6.8 911-933 AD-77162 51.6 5.4 93.6 3.1 2267-2289 AD-77163 41.24.4 70 3.1 2277-2299 AD-77164 58.5 5.8 95.5 10.9 2270-2292 AD-77165 11.12 61.7 7.1 6796-6818 AD-77166 40.3 5.6 74.2 1.7 2298-2320 AD-77167 559.2 84.6 18 2835-2857 AD-77168 7.9 0.6 28.4 3.6 7184-7206 AD-77169 36.17.5 51.6 5.6 1075-1097 AD-77170 44.4 8.8 66.7 3.6 1572-1594 AD-7717113.1 1.6 50.1 9.3 7212-7234 AD-77172 38.8 5.4 103.7 12.7 6826-6848AD-77173 42.4 2.8 79.8 15.2 3205-3227 AD-77174 48.9 3.5 83.3 6.51371-1393 Mock 100 6.1 100 5.6

TABLE 17 Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAsSense SEQ Antisense SEQ SEQ Duplex Oligo ID Oligo ID ID Name NameSense sequence NO Name Antisense sequence NO mRNA target sequence NOAD-75740 A-151647 asasuguaAfcUfAfAfuugaaucauaL96 1861 A-151648VPusAfsugaUfuCfAfauuaGfuUfacauususu 1954 AAAAUGUAACUAAUUGAAUCAUU 2047AD-75741 A-151649 usasagaaAfgUfAfCfuugacuaaaaL96 1862 A-151650VPusUfsuuaGfuCfAfaguaCfuUfucuuasasa 1955 UUUAAGAAAGUACUUGACUAAAA 2048AD-75747 A-151661 csusuuauAfaUfUfCfuugaaugagaL96 1863 A-151662VPusCfsucaUfuCfAfagaaUfuAfuaaagscsa 1956 UGCUUUAUAAUUCUUGAAUGAGG 2049AD-75748 A-151663 asgsauagAfaAfUfGfuauguuugaaL96 1864 A-151664VPusUfscaaAfcAfUfacauUfuCfuaucusasg 1957 CUAGAUAGAAAUGUAUGUUUGAC 2050AD-75749 A-151665 ususccacAfaUfCfCfuuagaaucuaL96 1865 A-151666VPusAfsgauUfcUfAfaggaUfuGfuggaasgsg 1958 CCUUCCACAAUCCUUAGAAUCUG 2051AD-75750 A-151667 usasucaaAfaCfCfUfuucaaauauaL96 1866 A-151668VPusAfsuauUfuGfAfaaggUfuUfugauasusu 1959 AAUAUCAAAACCUUUCAAAUAUC 2052AD-75751 A-151669 ascsaaguAfaAfCfAfuuccaacauaL96 1867 A-151670VPusAfsuguUfgGfAfauguUfuAfcuugusgsu 1960 ACACAAGUAAACAUUCCAACAUU 2053AD-75755 A-151677 ascsauagAfaAfGfUfuucuucaacaL96 1868 A-151678VPusGfsuugAfaGfAfaacuUfuCfuaugususu 1961 AAACAUAGAAAGUUUCUUCAACU 2054AD-75757 A-151681 csasgucaAfeAfAfGfuauuuuaacaL96 1869 A-151682VPusGfsuuaAfaAfUfacuuGfuUfgacugsasa 1962 UUCAGUCAACAAGUAUUUUAACU 2055AD-75759 A-151685 csuscaagCfuGfUfCfuacuuacauaL96 1870 A-151686VPusAfsuguAfaGfUfagacAfgCfuugagsgsu 1963 ACCUCAAGCUGUCUACUUACAUC 2056AD-75760 A-151687 gsasauugUfuUfCfCfuuauuugcaaL96 1871 A-151688VPusUfsgcaAfaUfAfaggaAfaCfaauucsasu 1964 AUGAAUUGUUUCCUUAUUUGCAC 2057AD-75761 A-151689 asuscuguCfuUfAfGfuugaaaagcaL96 1872 A-151690VPusGfscuuUfuCfAfacuaAfgAfcagausgsu 1965 ACAUCUGUCUUAGUUGAAAAGCA 2058AD-75765 A-151697 asasuuagCfaAfUfAfuauuauccaaL96 1873 A-151698VPusUfsggaUfaAfUfauauUfgCfuaauususu 1966 AAAAUUAGCAAUAUAUUAUCCAA 2059AD-75766 A-151699 asasuugaAfuCfAfUfuaucuuacaaL96 1874 A-151700VPusUfsguaAfgAfUfaaugAfuUfcaauusasg 1967 CUAAUUGAAUCAUUAUCUUACAU 2060AD-75769 A-151705 ususuaucAfaUfAfAfuguucuauaaL96 1875 A-151706VPusUfsauaGfaAfCfauuaUfuGfauaaasasg 1968 CUUUUAUCAAUAAUGUUCUAUAG 2061AD-75772 A-151711 gsusaaaaGfaAfAfCfuauacaucaaL96 1876 A-151712VPusUfsgauGfuAfUfaguuUfcUfuuuacsasu 1969 AUGUAAAAGAAACUAUACAUCAU 2062AD-75774 A-151715 asasugagGfaAfUfAfauaaguuaaaL96 1877 A-151716VPusUfsuaaCfuUfAfuuauUfcCfucauuscsu 1970 AGAAUGAGGAAUAAUAAGUUAAA 2063AD-75776 A-151719 csuscuguGfaAfUfGfuguuuuaucaL96 1878 A-151720VPusGfsauaAfaAfCfacauUfcAfcagagsasg 1971 CUCUCUGUGAAUGUGUUUUAUCC 2064AD-75778 A-151723 gsusuccuUfcAfAfAfugaugaguuaL96 1879 A-151724VPusAfsacuCfaUfCfauuuGfaAfggaacsusc 1972 GAGUUCCUUCAAAUGAUGAGUUA 2065AD-75779 A-151725 csasggauAfaAfGfAfuaucaauuuaL96 1880 A-151726VPusAfsaauUfgAfUfaucuUfuAfuccugsusa 1973 UACAGGAUAAAGAUAUCAAUUUA 2066AD-75787 A-151741 csasugucCfuCfCfUfcgcaucucuaL96 1881 A-151742VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu 1974 ACCAUGUCCUCCUCGCAUCUCUU 2067AD-75787 A-151741 csasugucCfuCfCfUfcgcaucucuaL96 1882 A-151742VPusAfsgagAfuGfCfgaggAfgGfacaugsgsu 1975 ACCAUGUCCUCCUCGCAUCUCUU 2068AD-75788 A-151743 ususcaacAfaGfCfCfcacaggguaaL96 1883 A-151744VPusUfsaccCfuGfUfgggcUfuGfuugaasasu 1976 AUUUCAACAAGCCCACAGGGUAU 2069AD-75788 A-151743 ususcaacAfaGfCfCfcacaggguaaL96 1884 A-151744VPusUfsaccCfuGfUfgggcUfuGfuugaasasu 1977 AUUUCAACAAGCCCACAGGGUAU 2070AD-75789 A-151745 uscscucgCfaUfCfUfcuucuaccuaL96 1885 A-151746VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg 1978 CCUCCUCGCAUCUCUUCUACCUG 2071AD-75789 A-151745 uscscucgCfaUfCfUfcuucuaccuaL96 1886 A-151746VPusAfsgguAfgAfAfgagaUfgCfgaggasgsg 1979 CCUCCUCGCAUCUCUUCUACCUG 2072AD-75790 A-151747 gsgsggcuUfuUfAfUfuucaacaagaL96 1887 A-151748VPusCfsuugUfuGfAfaauaAfaAfgccccsusg 1980 CAGGGGCUUUUAUUUCAACAAGC 2073AD-75790 A-151747 gsgsggcuUfuUfAfUfuucaacaagaL96 1888 A-151748VPusCfsuugUfuGfAfaauaAfaAfgccecsusg 1981 CAGGGGCUUUUAUUUCAACAAGC 2074AD-75791 A-151749 gsasugcuCfuUfCfAfguucgugugaL96 1889 A-151750VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa 1982 UGGAUGCUCUUCAGUUCGUGUGU 2075AD-75791 A-151749 gsasugcuCfuUfCfAfguucgugugaL96 1890 A-151750VPusCfsacaCfgAfAfcugaAfgAfgcaucscsa 1983 UGGAUGCUCUUCAGUUCGUGUGU 2076AD-75792 A-151751 usgsgaugCfuCfUfUfcaguucgugaL96 1891 A-151752VPusCfsacgAfaCfUfgaagAfgCfauccascsc 1984 GGUGGAUGCUCUUCAGUUCGUGU 2077AD-75792 A-151751 usgsgaugCfuCfUfUfcaguucgugaL96 1892 A-151752VPusCfsacgAfaCfUfgaagAfgCfauccascsc 1985 GGUGGAUGCUCUUCAGUUCGUGU 2078AD-75793 A-151753 gsgsuggaUfgCfUfCfuucaguucgaL96 1893 A-151754VPusCfsgaaCfuGfAfagagCfaUfccaccsasg 1986 CUGGUGGAUGCUCUUCAGUUCGU 2079AD-75793 A-151753 gsgsuggaUfgCfUfCfuucaguucgaL96 1894 A-151754VPusCfsgaaCfuGfAfagagCfaUfccaccsasg 1987 CUGGUGGAUGCUCUUCAGUUCGU 2080AD-75794 A-151755 gscsugguGfgAfUfGfcucuucaguaL96 1895 A-151756VPusAfscugAfaGfAfgcauCfcAfccagcsusc 1988 GAGCUGGUGGAUGCUCUUCAGUU 2081AD-75794 A-151755 gscsugguGfgAfUfGfcucuucaguaL96 1896 A-151756VPusAfscugAfaGfAfgcauCfcAfccagcsusc 1989 GAGCUGGUGGAUGCUCUUCAGUU 2082AD-75795 A-151757 csusggugGfaUfGfCfucuucaguuaL96 1897 A-151758VPusAfsacuGfaAfGfagcaUfcCfaccagscsu 1990 AGCUGGUGGAUGCUCUUCAGUUC 2083AD-75795 A-151757 csusggugGfaUfGfCfucuucaguuaL96 1898 A-151758VPusAfsacuGfaAfGfagcaUfcCfaccagscsu 1991 AGCUGGUGGAUGCUCUUCAGUUC 2084AD-77120 A-154752 cscsaaaaUfgCfAfCfugauguaaaaL96 1899 A-154753VPusUfsuuaCfaUfCfagugCfaUfuuuggsgsc 1992 GCCCAAAAUGCACUGAUGUAAAG 2085AD-77121 A-154754 uscscaguUfgCfAfCfuaaauuccuaL96 1900 A-154755VPusAfsggaAfuUfUfagugCfaAfeuggasusc 1993 GAUCCAGUUGCACUAAAUUCCUC 2086AD-77122 A-154756 csasuucuUfcAfAfCfuaucuuugaaL96 1901 A-154757VPusUfscaaAfgAfUfaguuGfaAfgaaugsasg 1994 CUCAUUCUUCAACUAUCUUUGAU 2087AD-77123 A-154758 ascsauucCfaAfCfAfuugucuuuaaL96 1902 A-154759VPusUfsaaaGfaCfAfauguUfgGfaaugususu 1995 AAACAUUCCAACAUUGUCUUUAG 2088AD-77124 A-154760 gsgscuuaGfaAfUfAfaaagauguaaL96 1903 A-154761VPusUfsacaUfcUfUfuuauUfcUfaagccsusu 1996 AAGGCUUAGAAUAAAAGAUGUAG 2089AD-77125 A-154762 ususucaaGfaUfAfUfuuguaaaagaL96 1904 A-154763VPusCfsuuuUfaCfAfaauaUfcUfugaaasusu 1997 AAUUUCAAGAUAUUUGUAAAAGA 2090AD-77126 A-154764 asusaagcAfuAfUfUfuugaaaaugaL96 1905 A-154765VPusCfsauuUfuCfAfaaauAfuGfcuuaususa 1998 UAAUAAGCAUAUUUUGAAAAUGU 2091AD-77127 A-154766 gsusuuucAfaUfGfCfuaguguuuaaL96 1906 A-154767VPusUfsaaaCfaCfUfagcaUfuGfaaaacsasa 1999 UUGUUUUCAAUGCUAGUGUUUAA 2092AD-77128 A-154768 csasaugaAfaUfAfCfacaaguaaaaL96 1907 A-154769VPusUfsuuaCfuUfGfuguaUfuUfcauugsgsg 2000 CCCAAUGAAAUACACAAGUAAAC 2093AD-77129 A-154770 csasaaagUfcCfAfCfugaugcaaaaL96 1908 A-154771VPusUfsuugCfaUfCfagugGfaCfuuuugsusg 2001 CACAAAAGUCCACUGAUGCAAAU 2094AD-77130 A-154772 asascauaGfaAfAfGfuuucuucaaaL96 1909 A-154773VPusUfsugaAfgAfAfacuuUfcUfauguususa 2002 UAAACAUAGAAAGUUUCUUCAAC 2095AD-77131 A-154774 asasccuaAfuUfAfGfuaacuuuccaL96 1910 A-154775VPusGfsgaaAfgUfUfacuaAfuUfagguusgsc 2003 GCAACCUAAUUAGUAACUUUCCU 2096AD-77132 A-154776 gsascuuaUfuUfCfCfuucacuuaaaL96 1911 A-154777VPusUfsuaaGfuGfAfaggaAfaUfaagucsasu 2004 AUGACUUAUUUCCUUCACUUAAU 2097AD-77133 A-154778 gsgscucuCfaAfAfCfuuagcaaaaaL96 1912 A-154779VPusUfsuuuGfcUfAfaguuUfgAfgagccsasa 2005 UUGGCUCUCAAACUUAGCAAAAU 2098AD-77134 A-154780 ascsaaagAfuAfAfGfugaaaagagaL96 1913 A-154781VPusCfsucuUfuUfCfacuuAfuCfuuugusasu 2006 AUACAAAGAUAAGUGAAAAGAGA 2099AD-77135 A-154782 gsusagugUfaCfUfGfuucaccaaanL96 1914 A-154783VPusUfsuugGfuGfAfacagUfaCfacuacsusu 2007 AAGUAGUGUACUGUUCACCAAAU 2100AD-77136 A-154784 uscsaccaAfcUfCfAfuagcaaaguaL96 1915 A-154785VPusAfscuuUfgCfUfaugaGfuUfggugasgsu 2008 ACUCACCAACUCAUAGCAAAGUC 2101AD-77137 A-154786 asasaauaAfcUfCfAfuuauaccaaaL96 1916 A-154787VPusUfsuggUfaUfAfaugaGfuUfauuuuscsa 2009 UGAAAAUAACUCAUUAUACCAAU 2102AD-77138 A-154788 asasgaaaGfaAfUfCfuuuccauucaL96 1917 A-154789VPusGfsaauGfgAfAfagauUfcUfuucuuscsu 2010 AGAAGAAAGAAUCUUUCCAUUCA 2103AD-77139 A-154790 asusuucaAfgAfUfAfuuuguaaaaaL96 1918 A-154791VPusUfsuuuAfcAfAfauauCfuUfgaaaususg 2011 CAAUUUCAAGAUAUUUGUAAAAG 2104AD-77140 A-154792 asgscugaAfaCfUfCfuagaauuaaaL96 1919 A-154793VPusUfsuaaUfuCfUfagagUfuUfcagcususg 2012 CAAGCUGAAACUCUAGAAUUAAA 2105AD-77141 A-154794 asasagauAfaAfUfCfugauuuaugaL96 1920 A-154795VPusCfsauaAfaUfCfagauUfuAfucuuususg 2013 CAAAAGAUAAAUCUGAUUUAUGC 2106AD-77142 A-154796 usasuuaaAfuUfAfAfuucugauugaL96 1921 A-154797VPusCfsaauCfaGfAfauuaAfuUfuaauasasa 2014 UUUAUUAAAUUAAUUCUGAUUGU 2107AD-77143 A-154798 asasgaauGfaGfCfUfuucaacucaaL96 1922 A-154799VPusUfsgagUfuGfAfaagcUfcAfuucuusasc 2015 GUAAGAAUGAGCUUUCAACUCAU 2108AD-77144 A-154800 gsusuugaUfaAfCfAfuuuaaaagaaL96 1923 A-154801VPusUfscuuUfuAfAfauguUfaUfcaaacsusu 2016 AAGUUUGAUAACAUUUAAAAGAU 2109AD-77145 A-154802 usgsuuuuAfaAfCfAfuagaaaguuaL96 1924 A-154803VPusAfsacuUfuCfUfauguUfuAfaaacasusa 2017 UAUGUUUUAAACAUAGAAAGUUU 2110AD-77146 A-154804 usasaauaAfcCfCfAfuucauagcaaL96 1925 A-154805VPusUfsgcuAfuGfAfauggGfuUfauuuasusa 2018 UAUAAAUAACCCAUUCAUAGCAU 2111AD-77147 A-154806 csascauaGfaCfUfCfuuuaaaacuaL96 1926 A-154807VPusAfsguuUfuAfAfagagUfcUfaugugsgsg 2019 CCCACAUAGACUCUUUAAAACUA 2112AD-77148 A-154808 asasgucaCfaAfAfGfuuaucuucuaL96 1927 A-154809VPusAfsgaaGfaUfAfacuuUfgUfgacuususu 2020 AAAAGUCACAAAGUUAUCUUCUU 2113AD-77149 A-154810 usasaagaAfaGfAfAfuuuaugagaaL96 1928 A-154811VPusUfscucAfuAfAfauucUfuUfcuuuasusc 2021 GAUAAAGAAAGAAUUUAUGAGAA 2114AD-77150 A-154812 gsasaaugUfaUfGfUfuugacuuguaL96 1929 A-154813VPusAfscaaGfuCfAfaacaUfaCfauuucsusa 2022 UAGAAAUGUAUGUUUGACUUGUU 2115AD-77151 A-154814 ususuauaAfgA1tfCfuuccuguuaaL96 1930 A-154815VPusUfsaacAfgGfAfagguCfuUfauaaasasu 2023 AUUUUAUAAGACCUUCCUGUUAG 2116AD-77152 A-154816 csusgugaAfuGfUfGfuuuuauccaaL96 1931 A-154817VPusUfsggaUfaAfAfacacAfuUfcacagsasg 2024 CUCUGUGAAUGUGUUUUAUCCAU 2117AD-77153 A-154818 ususaauuCfuGfAfUfuguauuugaaL96 1932 A-154819VPusUfscaaAfuAfCfaaucAfgAfauuaasusu 2025 AAUUAAUUCUGAUUGUAUUUGAA 2118AD-77154 A-154820 usgsucauCfuAfCfCfuaccucaaaaL96 1933 A-154821VPusUfsuugAfgGfUfagguAfgAfugacasusa 2026 UAUGUCAUCUACCUACCUCAAAG 2119AD-77155 A-154822 csgsugaaAfgCfAfAfaacaauaggaL96 1934 A-154823VPusCfscuaUfuGfUfuuugCfuUfucacgsusa 2027 UACGUGAAAGCAAAACAAUAGGG 2120AD-77156 A-154824 asascaacAfaUfUfCfuauagauagaL96 1935 A-154825VPusCfsuauCfuAfUfagaaUfuGfuuguususu 2028 AAAACAACAAUUCUAUAGAUAGA 2121AD-77157 A-154826 usasaaguAfuUfAfUfuugaacuuuaL96 1936 A-154827VPusAfsaagUfuCfAfaauaAfuAfcuuuasasu 2029 AUUAAAGUAUUAUUUGAACUUUU 2122AD-77158 A-154828 asgsaaagAfaUfUfUfaugagaaauaL96 1937 A-154829VPusAfsuuuCfuCfAfuaaaUfuCfuuucususu 2030 AAAGAAAGAAUUUAUGAGAAAUU 2123AD-77159 A-154830 asasaugcAfcUfGfAfuguaaaguaaL96 1938 A-154831VPusUfsacuUfuAfCfaucaGfuGfcauuususg 2031 CAAAAUGCACUGAUGUAAAGUAG 2124AD-77160 A-154832 asusuaauUfcUfGfAfuuguauuugaL96 1939 A-154833VPusCfsaaaUfaCfAfaucaGfaAfuuaaususu 2032 AAAUUAAUUCUGAUUGUAUUUGA 2125AD-77161 A-154834 csasauaaUfgUfUfCfuauagaaaaaL96 1940 A-154835VPusUfsuuuCfuAfUfagaaCfaUfuauugsasu 2033 AUCAAUAAUGUUCUAUAGAAAAG 2126AD-77162 A-154836 gsasuuuuCfaAfUfUfugauuuugaaL96 1941 A-154837VPusUfscaaAfaUfCfaaauUfgAfaaaucsasa 2034 UUGAUUUUCAAUUUGAUUUUGAA 2127AD-77163 A-154840 ususgauuUfuGfAfAfuucugcauuaL96 1942 A-154841VPusAfsaugCfaGfAfauucAfaAfaucaasasu 2035 AUUUGAUUUUGAAUUCUGCAUUU 2128AD-77164 A-154842 ususucaaUfuUfGfAfuuuugaauuaL96 1943 A-154843VPusAfsauuCfaAfAfaucaAfaUfugaaasasu 2036 AUUUUCAAUUUGAUUUUGAAUUC 2129AD-77165 A-154844 gsusgucuGfuGfUfAfucaugaaaaaL96 1944 A-154845VPusUfsuuuCfaUfGfauacAfcAfgacacsasg 2037 CUGUGUCUGUGUAUCAUGAAAAU 2130AD-77166 A-154846 gsgsuuuuAfuGfAfAfuacaaagauaL96 1945 A-154847VPusAfsucuUfuGfUfauucAfuAfaaaccsasa 2038 UUGGUUUUAUGAAUACAAAGAUA 2131AD-77167 A-154848 gscsagauCfaAfGfAfuuuucucauaL96 1946 A-154849VPusAfsugaGfaAfAfaucuUfgAfucugcsasg 2039 CUGCAGAUCAAGAUUUUCUCAUU 2132AD-77168 A-154850 asascuaaUfuGfAfAfucauuaucuaL96 1947 A-154851VPusAfsgauAfaUfGfauucAfaUfuaguusasc 2040 GUAACUAAUUGAAUCAUUAUCUU 2133AD-77169 A-154852 usgsguuuAfuGfAfAfuuguuuccuaL96 1948 A-154853VPusAfsggaAfaCfAfauucAfuAfaaccascsu 2041 AGUGGUUUAUGAAUUGUUUCCUU 2134AD-77170 A-154854 usasguauAfaUfGfGfugcuauuuuaL96 1949 A-154855VPusAfsaaaUfaGfCfaccaUfuAfuacuasasa 2042 UUUAGUAUAAUGGUGCUAUUUUG 2135AD-77171 A-154856 csusguuuAfaUfAfAfgcauauuuuaL96 1950 A-154857VPusAfsaaaUfaUfGfcuuaUfuAfaacagsusa 2043 UACUGUUUAAUAAGCAUAUUUUG 2136AD-77172 A-154858 asasauauCfaAfAfAfccuuucaaaaL96 1951 A-154859VPusUfsuugAfaAfGfguuuUfgAfuauuususg 2044 CAAAAUAUCAAAACCUUUCAAAU 2137AD-77173 A-154860 ascsagauGfuAfAfAfagaaacuauaL96 1952 A-154861VPusAfsuagUfuUfCfuuuuAfcAfucuguscsc 2045 GGACAGAUGUAAAAGAAACUAUA 2138AD-77174 A-154862 asascuuuGfaGfGfCfcaaucauuuaL96 1953 A-154863VPusAfsaauGfaUfUfggccUfcAfaaguusgsc 2046 GCAACUUUGAGGCCAAUCAUUUU 2139

Example 11-Further IGF-1 Transcripts, siRNA Design, and siRNA Screening

Bioinformatics:

A set of siRNAs targeting human IGF1 (human insulin like growth factor1, NCBI refseqID: NM_000618; NCBI GeneID: 3479) were designed usingcustom R and Python scripts. The human IGF1 REFSEQ mRNA has a length of7366 bases.

The rationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer siRNA from position 10through the end was determined with a linear model derived the directmeasure of mRNA knockdown from more than 20,000 distinct siRNA designstargeting a large number of vertebrate genes. The custom Python scriptbuilt the set of siRNAs by systematically selecting a siRNA every 11bases along the target mRNA starting at position 10. At each of thepositions, the neighboring siRNA (one position to the 5′ end of themRNA, one position to the 3′ end of the mRNA) was swapped into thedesign set if the predicted efficacy was better than the efficacy at theexact every-11th siRNA. Low complexity siRNAs, i.e., those with ShannonEntropy measures below 1.35 were excluded from the set.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,before being released from the plate by trypsinization. Three humanIGF-1 Dual-Glo® Luciferase constructs were generated using the psiCHECK2vector. Construct one contained sequence based on NM_001111285, whileconstructs two and three contained sequence based on NM_000618 asprovided in the prior Example. Dual-luciferase plasmids wereco-transfected with siRNA into 5000 cells using Lipofectamine RNAiMax(Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 384well plate, 0.1 μl of Lipofectamine was added to 15 ng of plasmid vectorand siRNA in 15 μl of Opti-MEM and allowed to complex at roomtemperature for 15 minutes. The mixture was then added to the cellsresuspended in 35 ul of fresh complete media. Cells were incubated for48 hours before luciferase was measured. Single dose experiments wereperformed at 10 nM final duplex concentration.

Dual-Glo® Luciferase Assay

Forty-eight hours after the siRNAs were transfected, Firefly(transfection control) and Renilla (fused to IGF1 target sequence in 3′UTR) luciferase were measured. First, media was removed from cells. ThenFirefly luciferase activity was measured by adding 20 ul of Dual-Glo®Luciferase Reagent mixed with 20 ul of complete media to each well. Themixture was incubated at room temperature for 30 minutes beforeluminescense (500 nm) was measured on a Spectramax (Molecular Devices)to detect the Firefly luciferase signal. Renilla luciferase activity wasmeasured by adding 20 ul of room temperature of Dual-Glo® Stop & Glo®Reagent to each well and the plates were incubated for 20 minutes beforeluminescence was again measured to determine the Renilla luciferasesignal. The Dual-Glo® Stop & Glo® Reagent quenched the fireflyluciferase signal and sustained luminescence for the Renilla luciferasereaction. siRNA activity was determined by normalizing the Renilla(IGF1) signal to the Firefly (control) signal within each well. Themagnitude of siRNA activity was then assessed relative to cells thatwere transfected with the same vector but were not treated with siRNA orwere treated with a non-targeting siRNA. All transfections were done inquadruplicates.

TABLE 18 Unmodified Sense and Antisense Strand Sequences of IGF-1 dsRNAsSense Position SEQ Antisense Position SEQ Duplex Oligo in NM_ ID Oligoin NM_ ID Name Name Sense Sequence 000618.3 NO Name Antisense Sequence000618.3 NO AD-74963 A-150432 UAGAUAAAUGUGAGGAUUU    6-24 2140 A-150433AAAUCCUCACAUUUAUCUA    6-24 2420 AD-74964 A-150434 UUCUCUAAAUCCCUCUUCU  24-42 2141 A-150435 AGAAGAGGGAUUUAGAGAA   24-42 2421 AD-74965 A-150436CUGUUUGCUAAAUCUCACU   41-59 2142 A-150437 AGUGAGAUUUAGCAAACAG   41-592422 AD-74966 A-150438 CUCACUGUCACUGCUAAAU   54-72 2143 A-150439AUUUAGCAGUGACAGUGAG   54-72 2423 AD-74967 A-150440 UUCAGAGCAGAUAGAGCCU  72-90 2144 A-150441 AGGCUCUAUCUGCUCUGAA   72-90 2424 AD-74968 A-150442CAUUGCUCUCAACAUCUCA  127-145 2145 A-150443 UGAGAUGUUGAGAGCAAUG  127-1452425 AD-74969 A-150444 ACCAAUUCAUUUUCAGACU  185-203 2146 A-150445AGUCUGAAAAUGAAUUGGU  185-203 2426 AD-74970 A-150446 UUUGUACUUCAGAAGCAAU 203-221 2147 A-150447 AUUGCUUCUGAAGUACAAA  203-221 2427 AD-74971A-150448 AUGGGAAAAAUCAGCAGUA  220-238 2148 A-150449 UACUGCUGAUUUUUCCCAU 220-238 2428 AD-74972 A-150450 CAAUUAUUUAAGUGCUGCU  247-265 2149A-150451 AGCAGCACUUAAAUAAUUG  247-265 2429 AD-74973 A-150452UUGAAGGUGAAGAUGCACA  277-295 2150 A-150453 UGUGCAUCUUCACCUUCAA  277-2952430 AD-74974 A-150454 UUUUAUUUCAACAAGCCCA  430-448 2151 A-150455UGGGCUUGUUGAAAUAAAA  430-448 2431 AD-74975 A-150456 CACAGGGUAUGGCUCCAGA 447-465 2152 A-150457 UCUGGAGCCAUACCCUGUG  447-465 2432 AD-74976A-150458 CAGCAGUCGGAGGGCGCCU  462-480 2153 A-150459 AGGCGCCCUCCGACUGCUG 462-480 2433 AD-74977 A-150460 UUGCGCACCCCUCAAGCCU  543-561 2154A-150461 AGGCUUGAGGGGUGCGCAA  543-561 2434 AD-74978 A-150462UGCAGGAAACAAGAACUAA  654-672 2155 A-150463 UUAGUUCUUGUUUCCUGCA  654-6722435 AD-74979 A-150464 CAGGAUGUAGGAAGACCCU  672-690 2156 A-150465AGGGUCUUCCUACAUCCUG  672-690 2436 AD-74980 A-150466 UUAAACUUUGGAACACCUA 750-768 2157 A-150467 UAGGUGUUCCAAAGUUUAA  750-768 2437 AD-74981A-150468 AAAUAAGUUUGAUAACAUU  774-792 2158 A-150469 AAUGUUAUCAAACUUAUUU 774-792 2438 AD-74982 A-150470 UUAAAAGAUGGGCGUUUCA  792-810 2159A-150471 UGAAACGCCCAUCUUUUAA  792-810 2439 AD-74983 A-150472AAAUACACAAGUAAACAUU  818-836 2160 A-150473 AAUGUUUACUUGUGUAUUU  818-8362440 AD-74984 A-150474 UUCCAACAUUGUCUUUAGA  835-853 2161 A-150475UCUAAAGACAAUGUUGGAA  835-853 2441 AD-74985 A-150476 GGAGUGAUUUGCACCUUGA 852-870 2162 A-150477 UCAAGGUGCAAAUCACUCC  852-870 2442 AD-74986A-150478 AUUGCUGUUGAUCUUUUAU  894-912 2163 A-150479 AUAAAAGAUCAACAGCAAU 894-912 2443 AD-74987 A-150480 UCAAUAAUGUUCUAUAGAA  912-930 2164A-150481 UUCUAUAGAACAUUAUUGA  912-930 2444 AD-74988 A-150482AAAGAAAAAAAAAAUAUAU  930-948 2165 A-150483 AUAUAUUUUUUUUUUCUUU  930-9482445 AD-74989 A-150484 AUAUAUAUAUAUAUCUUAA  947-965 2166 A-150485UUAAGAUAUAUAUAUAUAU  947-965 2446 AD-74990 A-150486 UUUCCUUAUUUGCACUUCU1091-1109 2167 A-150487 AGAAGUGCAAAUAAGGAAA 1091-1109 2447 AD-74991A-150488 CUUUCUACACAACUCGGGA 1108-1126 2168 A-150489 UCCCGAGUUGUGUAGAAAG1108-1126 2448 AD-74992 A-150490 GCUGUUUGUUUUACAGUGU 1125-1143 2169A-150491 ACACUGUAAAACAAACAGC 1125-1143 2449 AD-74993 A-150492UUACAGUGUCUGAUAAUCU 1135-1153 2170 A-150493 AGAUUAUCAGACACUGUAA1135-1153 2450 AD-74994 A-150494 CUGAUAAUCUUGUUAGUCU 1144-1162 2171A-150495 AGACUAACAAGAUUAUCAG 1144-1162 2451 AD-74995 A-150496UAUACCCACCACCUCCCUU 1162-1180 2172 A-150497 AAGGGAGGUGGUGGGUAUA1162-1180 2452 AD-74996 A-150498 UUGCCGAAUUUGGCCUCCU 1195-1213 2173A-150499 AGGAGGCCAAAUUCGGCAA 1195-1213 2453 AD-74997 A-150500GCCGAAUUUGGCCUCCUCA 1197-1215 2174 A-150501 UGAGGAGGCCAAAUUCGGC1197-1215 2454 AD-74998 A-150502 AAAAGCAGCAGCAAGUCGU 1215-1233 2175A-150503 ACGACUUGCUGCUGCUUUU 1215-1233 2455 AD-74999 A-150504GUCAAGAAGCACACCAAUU 1232-1250 2176 A-150505 AAUUGGUGUGCUUCUUGAC1232-1250 2456 AD-75000 A-150506 AGUUGGAUGCAUUUUAUUU 1293-1311 2177A-150507 AAAUAAAAUGCAUCCAACU 1293-1311 2457 AD-75001 A-150508UUAGACACAAAGCUUUAUU 1311-1329 2178 A-150509 AAUAAAGCUUUGUGUCUAA1311-1329 2458 AD-75002 A-150510 CACAUCAUGCUUACAAAAA 1334-1352 2179A-150511 UUUUUGUAAGCAUGAUGUG 1334-1352 2459 AD-75003 A-150512AAGAAUAAUGCAAAUAGUU 1352-1370 2180 A-150513 AACUAUUUGCAUUAUUCUU1352-1370 2460 AD-75004 A-150514 UGCAACUUUGAGGCCAAUA 1370-1388 2181A-150515 UAUUGGCCUCAAAGUUGCA 1370-1388 2461 AD-75005 A-150516CAUUUUUAGGCAUAUGUUU 1388-1406 2182 A-150517 AAACAUAUGCCUAAAAAUG1388-1406 2462 AD-75006 A-150518 UUAAACAUAGAAAGUUUCU 1406-1424 2183A-150519 AGAAACUUUCUAUGUUUAA 1406-1424 2463 AD-75007 A-150520CUUCAACUCAAAAGAGUUA 1423-1441 2184 A-150521 UAACUCUUUUGAGUUGAAG1423-1441 2464 AD-75008 A-150522 UCCUUCAAAUGAUGAGUUA 1440-1458 2185A-150523 UAACUCAUCAUUUGAAGGA 1440-1458 2465 AD-75009 A-150524UUAGUAACUUUCCUCUUUU 1472-1490 2186 A-150525 AAAAGAGGAAAGUUACUAA1472-1490 2466 AD-75010 A-150526 UUUUUCCAUAUAGAGCACU 1494-1512 2187A-150527 AGUGCUCUAUAUGGAAAAA 1494-1512 2467 AD-75011 A-150528CUAUGUAAAUUUAGCAUAU 1511-1529 2188 A-150529 AUAUGCUAAAUUUACAUAG1511-1529 2468 AD-75012 A-150530 AUCAAUUAUACAGGAUAUA 1528-1546 2189A-150531 UAUAUCCUGUAUAAUUGAU 1528-1546 2469 AD-75013 A-150532UUUAGUAUAAUGGUGCUAU 1572-1590 2190 A-150533 AUAGCACCAUUAUACUAAA1572-1590 2470 AD-75014 A-150534 UUGUUAUAUGAAAGAGUCU 1599-1617 2191A-150535 AGACUCUUUCAUAUAACAA 1599-1617 2471 AD-75015 A-150536ACGGUAAUACGUGAAAGCA 1625-1643 2192 A-150537 UGCUUUCACGUAUUACCGU1625-1643 2472 AD-75016 A-150538 AAAACAAUAGGGGAAGCCU 1643-1661 2193A-150539 AGGCUUCCCCUAUUGUUUU 1643-1661 2473 AD-75017 A-150540UACUGAAAACACCAUCCAU 1690-1708 2194 A-150541 AUGGAUGGUGUUUUCAGUA1690-1708 2474 AD-75018 A-150542 UUGGGAAAGAAGGCAAAGU 1709-1727 2195A-150543 ACUUUGCCUUCUUUCCCAA 1709-1727 2475 AD-75019 A-150544UCAGACACAAAAGUCCACU 1757-1775 2196 A-150545 AGUGGACUUUUGUGUCUGA1757-1775 2476 AD-75020 A-150546 CGAGUCCAGAGAGGAAACU 1793-1811 2197A-150547 AGUUUCCUCUCUGGACUCG 1793-1811 2477 AD-75021 A-150548AAACUGUGGAAUGGAAAAA 1807-1825 2198 A-150549 UUUUUCCAUUCCACAGUUU1807-1825 2478 AD-75022 A-150550 AGCAGAAGGCUAGGAAUUU 1825-1843 2199A-150551 AAAUUCCUAGCCUUCUGCU 1825-1843 2479 AD-75023 A-150552UUAGCAGUCCUGGUUUCUU 1843-1861 2200 A-150553 AAGAAACCAGGACUGCUAA1843-1861 2480 AD-75024 A-150554 CAAAAUGGGGGCAAUAUGU 1966-1984 2201A-150555 ACAUAUUGCCCCCAUUUUG 1966-1984 2481 AD-75025 A-150556UUUAAAAAGAUAAAGAUUA 2016-2034 2202 A-150557 UAAUCUUUAUCUUUUUAAA2016-2034 2482 AD-75026 A-150558 UCAGAUUUUUUUUACCCUA 2033-2051 2203A-150559 UAGGGUAAAAAAAAUCUGA 2033-2051 2483 AD-75027 A-150560UUUUUUACCCUGGGUUGCU 2040-2058 2204 A-150561 AGCAACCCAGGGUAAAAAA2040-2058 2484 AD-75028 A-150562 CUGUAAGGGUGCAACAUCA 2057-2075 2205A-150563 UGAUGUUGCACCCUUACAG 2057-2075 2485 AD-75029 A-150564CUGAGAUGCAAGGAAUUCU 2090-2108 2206 A-150565 AGAAUUCCUUGCAUCUCAG2090-2108 2486 AD-75030 A-150566 UUGGUGAAUUGAAUGCUCA 2140-2158 2207A-150567 UGAGCAUUCAAUUCACCAA 2140-2158 2487 AD-75031 A-150568UUCUUGUCAGUGAAGCUAU 2170-2188 2208 A-150569 AUAGCUUCACUGACAAGAA2170-2188 2488 AD-75032 A-150570 AAUAACUGGCCAACUAGUU 2192-2210 2209A-150571 AACUAGUUGGCCAGUUAUU 2192-2210 2489 AD-75033 A-150572UGUUAAAAGCUAACAGCUA 2210-2228 2210 A-150573 UAGCUGUUAGCUUUUAACA2210-2228 2490 AD-75034 A-150574 CAAUCUCUUAAAACACUUU 2228-2246 2211A-150575 AAAGUGUUUUAAGAGAUUG 2228-2246 2491 AD-75035 A-150576AAAAUAUGUGGGAAGCAUU 2249-2267 2212 A-150577 AAUGCUUCCCACAUAUUUU2249-2267 2492 AD-75036 A-150578 UUUGAUUUUCAAUUUGAUU 2266-2284 2213A-150579 AAUCAAAUUGAAAAUCAAA 2266-2284 2493 AD-75037 A-150580UUGAAUUCUGCAUUUGGUU 2285-2303 2214 A-150581 AACCAAAUGCAGAAUUCAA2285-2303 2494 AD-75038 A-150582 UUUAUGAAUACAAAGAUAA 2303-2321 2215A-150583 UUAUCUUUGUAUUCAUAAA 2303-2321 2495 AD-75039 A-150584GUGAAAAGAGAGAAAGGAA 2322-2340 2216 A-150585 UUCCUUUCUCUCUUUUCAC2322-2340 2496 AD-75040 A-150586 AAAGAAAAAGGAGAAAAAC 2340-2358 2217A-150587 GUUUUUCUCCUUUUUCUUU 2340-2358 2497 AD-75041 A-150588ACAAAGAGAUUUCUACCAA 2357-2375 2218 A-150589 UUGGUAGAAAUCUCUUUGU2357-2375 2498 AD-75042 A-150590 UUGUUAGCACUCACUGACU 2398-2416 2219A-150591 AGUCAGUGAGUGCUAACAA 2398-2416 2499 AD-75043 A-150592UACAUAUCUAGUAAAACCU 2432-2450 2220 A-150593 AGGUUUUACUAGAUAUGUA2432-2450 2500 AD-75044 A-150594 CUCGUUUAAUACUAUAAAU 2449-2467 2221A-150595 AUUUAUAGUAUUAAACGAG 2449-2467 2501 AD-75045 A-150596UUUAAUACUAUAAAUAAUA 2453-2471 2222 A-150597 UAUUAUUUAUAGUAUUAAA2453-2471 2502 AD-75046 A-150598 UAUUCUAUUCAUUUUGAAA 2470-2488 2223A-150599 UUUCAAAAUGAAUAGAAUA 2470-2488 2503 AD-75047 A-150600UUUGAAAAACACAAUGAUU 2482-2500 2224 A-150601 AAUCAUUGUGUUUUUCAAA2482-2500 2504 AD-75048 A-150602 AAGGAAAGUGAUCCAAAAU 2521-2539 2225A-150603 AUUUUGGAUCACUUUCCUU 2521-2539 2505 AD-75049 A-150604UUUGAAAUAUUAAAAUAAU 2539-2557 2226 A-150605 AUUAUUUUAAUAUUUCAAA2539-2557 2506 AD-75050 A-150606 UUAAAAUAAUAUCUAAUAA 2548-2566 2227A-150607 UUAUUAGAUAUUAUUUUAA 2548-2566 2507 AD-75051 A-150608AAAAGUCACAAAGUUAUCU 2566-2584 2228 A-150609 AGAUAACUUUGUGACUUUU2566-2584 2508 AD-75052 A-150610 UUCUUUAACAAACUUUACU 2584-2602 2229A-150611 AGUAAAGUUUGUUAAAGAA 2584-2602 2509 AD-75053 A-150612CUCUUAUUCUUAGCUGUAU 2601-2619 2230 A-150613 AUACAGCUAAGAAUAAGAG2601-2619 2510 AD-75054 A-150614 AUAUACAUUUUUUUAAAAG 2618-2636 2231A-150615 CUUUUAAAAAAAUGUAUAU 2618-2636 2511 AD-75055 A-150616CAUUUUUUUAAAAGUUUGU 2623-2641 2232 A-150617 ACAAACUUUUAAAAAAAUG2623-2641 2512 AD-75056 A-150618 GUUAAAAUAUGCUUGACUA 2640-2658 2233A-150619 UAGUCAAGCAUAUUUUAAC 2640-2658 2513 AD-75057 A-150620AUGCUUGACUAGAGUUUCA 2648-2666 2234 A-150621 UGAAACUCUAGUCAAGCAU2648-2666 2514 AD-75058 A-150622 CAGUUGAAAGGCAAAAACU 2666-2684 2235A-150623 AGUUUUUGCCUUUCAACUG 2666-2684 2515 AD-75059 A-150624UUCCAUCACAACAAGAAAU 2684-2702 2236 A-150625 AUUUCUUGUUGUGAUGGAA2684-2702 2516 AD-75060 A-150626 UUGGUAUCAAGAAAGUCCA 2771-2789 2237A-150627 UGGACUUUCUUGAUACCAA 2771-2789 2517 AD-75061 A-150628GUUAGUGUACUAGUCCAUA 2793-2811 2238 A-150629 UAUGGACUAGUACACUAAC2793-2811 2518 AD-75062 A-150630 CAUAGCCUAGAAAAUGAUA 2811-2829 2239A-150631 UAUCAUUUUCUAGGCUAUG 2811-2829 2519 AD-75063 A-150632UCCCUAUCUGCAGAUCAAA 2828-2846 2240 A-150633 UUUGAUCUGCAGAUAGGGA2828-2846 2520 AD-75064 A-150634 UUAUCCAGCAUUCAGAUCU 2869-2887 2241A-150635 AGAUCUGAAUGCUGGAUAA 2869-2887 2521 AD-75065 A-150636UUUUUGGUUAAAAGUACCA 2906-2924 2242 A-150637 UGGUACUUUUAACCAAAAA2906-2924 2522 AD-75066 A-150638 UACCCAGGCUUGAUUAUUU 2920-2938 2243A-150639 AAAUAAUCAAGCCUGGGUA 2920-2938 2523 AD-75067 A-150640UCAUGCAAAUUCUAUAUUU 2938-2956 2244 A-150641 AAAUAUAGAAUUUGCAUGA2938-2956 2524 AD-75068 A-150642 UUACAUUCUUGGAAAGUCU 2956-2974 2245A-150643 AGACUUUCCAAGAAUGUAA 2956-2974 2525 AD-75069 A-150644UCUUGGAAAGUCUAUAUGA 2962-2980 2246 A-150645 UCAUAUAGACUUUCCAAGA2962-2980 2526 AD-75070 A-150646 AAAAACAAAAAUAACAUCU 2980-2998 2247A-150647 AGAUGUUAUUUUUGUUUUU 2980-2998 2527 AD-75071 A-150648UUCUCCCACUGGGUCACCU 3006-3024 2248 A-150649 AGGUGACCCAGUGGGAGAA3006-3024 2528 AD-75072 A-150650 CAAGGAUCAGAGGCCAGGA 3025-3043 2249A-150651 UCCUGGCCUCUGAUCCUUG 3025-3043 2529 AD-75073 A-150652AAAAAAAAAAAAAAGACUA 3043-3061 2250 A-150653 UAGUCUUUUUUUUUUUUUU3043-3061 2530 AD-75074 A-150654 UCCCUGGAUCUCUGAAUAU 3060-3078 2251A-150655 AUAUUCAGAGAUCCAGGGA 3060-3078 2531 AD-75075 A-150656AUAUGCAAAAAGAAGGCCA 3077-3095 2252 A-150657 UGGCCUUCUUUUUGCAUAU3077-3095 2532 AD-75076 A-150658 UAGUGGAGCCAGCAAUCCU 3100-3118 2253A-150659 AGGAUUGCUGGCUCCACUA 3100-3118 2533 AD-75077 A-150660UUAACUCUCAGUCCAACAU 3137-3155 2254 A-150661 AUGUUGGACUGAGAGUUAA3137-3155 2534 AD-75078 A-150662 UUAUUUGAAUUGAGCACCU 3155-3173 2255A-150663 AGGUGCUCAAUUCAAAUAA 3155-3173 2535 AD-75079 A-150664CAGAUGUAAAAGAAACUAU 3208-3226 2256 A-150665 AUAGUUUCUUUUACAUCUG3208-3226 2536 AD-75080 A-150666 AUACAUCAUUUUUGCCCUA 3225-3243 2257A-150667 UAGGGCAAAAAUGAUGUAU 3225-3243 2537 AD-75081 A-150668UUUUGCCCUCUGCCUGUUU 3234-3252 2258 A-150669 AAACAGGCAGAGGGCAAAA3234-3252 2538 AD-75082 A-150670 UUCCAGACAUACAGGUUCU 3252-3270 2259A-150671 AGAACCUGUAUGUCUGGAA 3252-3270 2539 AD-75083 A-150672CUGUGGAAUAAGAUACUGA 3269-3287 2260 A-150673 UCAGUAUCUUAUUCCACAG3269-3287 2540 AD-75084 A-150674 UAAGAUACUGGACUCCUCU 3277-3295 2261A-150675 AGAGGAGUCCAGUAUCUUA 3277-3295 2541 AD-75085 A-150676CUUCCCAAGAUGGCACUUA 3294-3312 2262 A-150677 UAAGUGCCAUCUUGGGAAG3294-3312 2542 AD-75086 A-150678 GUGUACCUUUUAAAAUUAU 3334-3352 2263A-150679 AUAAUUUUAAAAGGUACAC 3334-3352 2543 AD-75087 A-150680UUCCCUCUCAACAAAACUU 3352-3370 2264 A-150681 AAGUUUUGUUGAGAGGGAA3352-3370 2544 AD-75088 A-150682 UUUAUAGGCAGUCUUCUGA 3369-3387 2265A-150683 UCAGAAGACUGCCUAUAAA 3369-3387 2545 AD-75089 A-150684UUUUCUGUCAUAGUUAGAU 3400-3418 2266 A-150685 AUCUAACUAUGACAGAAAA3400-3418 2546 AD-75090 A-150686 AUGUGAUAAUUCUAAGAGU 3417-3435 2267A-150687 ACUCUUAGAAUUAUCACAU 3417-3435 2547 AD-75091 A-150688UUCCUUCACUUAAUUCUAU 3449-3467 2268 A-150689 AUAGAAUUAAGUGAAGGAA3449-3467 2548 AD-75092 A-150690 AUUAUCUUUCUUAACUUUU 3517-3535 2269A-150691 AAAAGUUAAGAAAGAUAAU 3517-3535 2549 AD-75093 A-150692UUCCAACACAUAAUCCUCU 3535-3553 2270 A-150693 AGAGGAUUAUGUGUUGGAA3535-3553 2550 AD-75094 A-150694 AAAUAAAUUGAAAAUAACU 3567-3585 2271A-150695 AGUUAUUUUCAAUUUAUUU 3567-3585 2551 AD-75095 A-150696UCAUUAUACCAAUUCACUA 3585-3603 2272 A-150697 UAGUGAAUUGGUAUAAUGA3585-3603 2552 AD-75096 A-150698 AUUUUAUUUUUUAAUGAAU 3603-3621 2273A-150699 AUUCAUUAAAAAAUAAAAU 3603-3621 2553 AD-75097 A-150700UUAAAACUAGAAAACAAAU 3621-3639 2274 A-150701 AUUUGUUUUCUAGUUUUAA3621-3639 2554 AD-75098 A-150702 UUGAUUACUAUAUACUACA 3662-3680 2275A-150703 UGUAGUAUAUAGUAAUCAA 3662-3680 2555 AD-75099 A-150704AUGACUCAGAUUUCAUAGA 3686-3704 2276 A-150705 UCUAUGAAAUCUGAGUCAU3686-3704 2556 AD-75100 A-150706 AAAGGAGCAACCAAAAUGU 3704-3722 2277A-150707 ACAUUUUGGUUGCUCCUUU 3704-3722 2557 AD-75101 A-150708GUCACAACCCAAAACUUUA 3721-3739 2278 A-150709 UAAAGUUUUGGGUUGUGAC3721-3739 2558 AD-75102 A-150710 AAACUUUACAAGCUUUGCU 3732-3750 2279A-150711 AGCAAAGCUUGUAAAGUUU 3732-3750 2559 AD-75103 A-150712UUCAGAAUUAGAUUGCUUU 3750-3768 2280 A-150713 AAAGCAAUCUAAUUCUGAA3750-3768 2560 AD-75104 A-150714 UUAUAAUUCUUGAAUGAGA 3767-3785 2281A-150715 UCUCAUUCAAGAAUUAUAA 3767-3785 2561 AD-75105 A-150716UAAUUCUUGAAUGAGGCAA 3770-3788 2282 A-150717 UUGCCUCAUUCAAGAAUUA3770-3788 2562 AD-75106 A-150718 AUUUCAAGAUAUUUGUAAA 3788-3806 2283A-150719 UUUACAAAUAUCUUGAAAU 3788-3806 2563 AD-75107 A-150720AAGAACAGUAAACAUUGGU 3806-3824 2284 A-150721 ACCAAUGUUUACUGUUCUU3806-3824 2564 AD-75108 A-150722 UUUCAACUCAUAGGCUUAU 3835-3853 2285A-150723 AUAAGCCUAUGAGUUGAAA 3835-3853 2565 AD-75109 A-150724UUGACCAUACUGGAUACUU 3865-3883 2286 A-150725 AAGUAUCCAGUAUGGUCAA3865-3883 2566 AD-75110 A-150726 UUUAAGAUGAGGCAGUUCA 3939-3957 2287A-150727 UGAACUGCCUCAUCUUAAA 3939-3957 2567 AD-75111 A-150728CAUCAGAAUCCACUCUUCU 3982-4000 2288 A-150729 AGAAGAGUGGAUUCUGAUG3982-4000 2568 AD-75112 A-150730 UAGGGAUAUGAAAAUCUCU 4000-4018 2289A-150731 AGAGAUUUUCAUAUCCCUA 4000-4018 2569 AD-75113 A-150732UUCACCCUAAGGAUCCAAU 4081-4099 2290 A-150733 AUUGGAUCCUUAGGGUGAA4081-4099 2570 AD-75114 A-150734 AUGGAAUACUGAAAAGAAA 4098-4116 2291A-150735 UUUCUUUUCAGUAUUCCAU 4098-4116 2571 AD-75115 A-150736GAAUACUGAAAAGAAAUCA 4101-4119 2292 A-150737 UGAUUUCUUUUCAGUAUUC4101-4119 2572 AD-75116 A-150738 ACUUCCUUGAAAAUUUUAU 4119-4137 2293A-150739 AUAAAAUUUUCAAGGAAGU 4119-4137 2573 AD-75117 A-150740UUAAAAAACAAACAAACAA 4137-4155 2294 A-150741 UUGUUUGUUUGUUUUUUAA4137-4155 2574 AD-75118 A-150742 AAACAAAAAGCCUGUCCAA 4154-4172 2295A-150743 UUGGACAGGCUUUUUGUUU 4154-4172 2575 AD-75119 A-150744UUUGUGUAGAUGAAACCAU 4208-4226 2296 A-150745 AUGGUUUCAUCUACACAAA4208-4226 2576 AD-75120 A-150746 UUGGGAGAAGGCUUAGAAU 4271-4289 2297A-150747 AUUCUAAGCCUUCUCCCAA 4271-4289 2577 AD-75121 A-150748UAAAAGAUGUAGCACAUUU 4289-4307 2298 A-150749 AAAUGUGCUACAUCUUUUA4289-4307 2578 AD-75122 A-150750 UUAUUGUUUGGCCAGCUAU 4319-4337 2299A-150751 AUAGCUGGCCAAACAAUAA 4319-4337 2579 AD-75123 A-150752AUGCCAAUGUGGUGCUAUU 4336-4354 2300 A-150753 AAUAGCACCACAUUGGCAU4336-4354 2580 AD-75124 A-150754 AAUGUGGUGCUAUUGUUUA 4341-4359 2301A-150755 UAAACAAUAGCACCACAUU 4341-4359 2581 AD-75125 A-150756CUUUAAGAAAGUACUUGAA 4359-4377 2302 A-150757 UUCAAGUACUUUCUUAAAG4359-4377 2582 AD-75126 A-150758 CUAAAAAAAAAAGAAAAAA 4377-4395 2303A-150759 UUUUUUCUUUUUUUUUUAG 4377-4395 2583 AD-75127 A-150760AAGAAAAAAAAGAAAGCAU 4395-4413 2304 A-150761 AUGCUUUCUUUUUUUUCUU4395-4413 2584 AD-75128 A-150762 AUAGACAUAUUUUUUUAAA 4412-4430 2305A-150763 UUUAAAAAAAUAUGUCUAU 4412-4430 2585 AD-75129 A-150764UUAAAGUAUAAAAACAACA 4426-4444 2306 A-150765 UGUUGUUUUUAUACUUUAA4426-4444 2586 AD-75130 A-150766 CAAUUCUAUAGAUAGAUGA 4443-4461 2307A-150767 UCAUCUAUCUAUAGAAUUG 4443-4461 2587 AD-75131 A-150768GGCUUAAUAAAAUAGCAUU 4460-4478 2308 A-150769 AAUGCUAUUUUAUUAAGCC4460-4478 2588 AD-75132 A-150770 UAAUAAAAUAGCAUUAGGU 4464-4482 2309A-150771 ACCUAAUGCUAUUUUAUUA 4464-4482 2589 AD-75133 A-150772UAUCUAGCCACCACCACCU 4484-4502 2310 A-150773 AGGUGGUGGUGGCUAGAUA4484-4502 2590 AD-75134 A-150774 UUUAUCACUCACAAGUAGU 4511-4529 2311A-150775 ACUACUUGUGAGUGAUAAA 4511-4529 2591 AD-75135 A-150776GGCAGGAGUUGGAAAUUUU 4566-4584 2312 A-150777 AAAAUUUCCAACUCCUGCC4566-4584 2592 AD-75136 A-150778 UUUAAAGUUAGAAGGCUCA 4584-4602 2313A-150779 UGAGCCUUCUAACUUUAAA 4584-4602 2593 AD-75137 A-150780CCAUUGUUUUGUUGGCUCU 4601-4619 2314 A-150781 AGAGCCAACAAAACAAUGG4601-4619 2594 AD-75138 A-150782 UUAGCAAAAUUAGCAAUAU 4625-4643 2315A-150783 AUAUUGCUAAUUUUGCUAA 4625-4643 2595 AD-75139 A-150784AUAUUAUCCAAUCUUCUGA 4642-4660 2316 A-150785 UCAGAAGAUUGGAUAAUAU4642-4660 2596 AD-75140 A-150786 UUAUCCAAUCUUCUGAACU 4645-4663 2317A-150787 AGUUCAGAAGAUUGGAUAA 4645-4663 2597 AD-75141 A-150788AAGAGCAUGGAGAAUAAAC 4669-4687 2318 A-150789 GUUUAUUCUCCAUGCUCUU4669-4687 2598 AD-75142 A-150790 ACGCGGGAAAAAAGAUCUU 4686-4704 2319A-150791 AAGAUCUUUUUUCCCGCGU 4686-4704 2599 AD-75143 A-150792GAUCUUAUAGGCAAAUAGA 4699-4717 2320 A-150793 UCUAUUUGCCUAUAAGAUC4699-4717 2600 AD-75144 A-150794 AAGAAUUUAAAAGAUAAGU 4717-4735 2321A-150795 ACUUAUCUUUUAAAUUCUU 4717-4735 2601 AD-75145 A-150796GUAAGUUCCUUAUUGAUUU 4734-4752 2322 A-150797 AAAUCAAUAAGGAACUUAC4734-4752 2602 AD-75146 A-150798 UUUUGUGCACUCUGCUCUA 4751-4769 2323A-150799 UAGAGCAGAGUGCACAAAA 4751-4769 2603 AD-75147 A-150800AAACAGAUAUUCAGCAAGU 4770-4788 2324 A-150801 ACUUGCUGAAUAUCUGUUU4770-4788 2604 AD-75148 A-150802 UCAGCAAGUGGAGAAAAUA 4780-4798 2325A-150803 UAUUUUCUCCACUUGCUGA 4780-4798 2605 AD-75149 A-150804AAGAACAAAGAGAAAAAAU 4798-4816 2326 A-150805 AUUUUUUCUCUUUGUUCUU4798-4816 2606 AD-75150 A-150806 AUACAUAGAUUUACCUGCA 4815-4833 2327A-150807 UGCAGGUAAAUCUAUGUAU 4815-4833 2607 AD-75151 A-150808UUACCUGCAAAAAAUAGCU 4825-4843 2328 A-150809 AGCUAUUUUUUGCAGGUAA4825-4843 2608 AD-75152 A-150810 UUUAUAGAAGACAUUCUCA 4884-4902 2329A-150811 UGAGAAUGUCUUCUAUAAA 4884-4902 2609 AD-75153 A-150812AGACAUCUCAAAGAGCAGU 4911-4929 2330 A-150813 ACUGCUCUUUGAGAUGUCU4911-4929 2610 AD-75154 A-150814 UAUGAGAUGGGGGUUAUCU 4987-5005 2331A-150815 AGAUAACCCCCAUCUCAUA 4987-5005 2611 AD-75155 A-150816CUACUGAUAAAGAAAGAAU 5004-5022 2332 A-150817 AUUCUUUCUUUAUCAGUAG5004-5022 2612 AD-75156 A-150818 AAAGAAUUUAUGAGAAAUU 5016-5034 2333A-150819 AAUUUCUCAUAAAUUCUUU 5016-5034 2613 AD-75157 A-150820UAACAAUCUGUGAAGAUUU 5050-5068 2334 A-150821 AAAUCUUCACAGAUUGUUA5050-5068 2614 AD-75158 A-150822 UUUUACUUUAUACAGUCUU 5098-5116 2335A-150823 AAGACUGUAUAAAGUAAAA 5098-5116 2615 AD-75159 A-150824UUUAUGAAUUUCUUAAUGU 5115-5133 2336 A-150825 ACAUUAAGAAAUUCAUAAA5115-5133 2616 AD-75160 A-150826 UUAAUGUUCAAAAUGACUU 5127-5145 2337A-150827 AAGUCAUUUUGAACAUUAA 5127-5145 2617 AD-75161 A-150828UUCUUCUUUUUUUAUAUCA 5153-5171 2338 A-150829 UGAUAUAAAAAAAGAAGAA5153-5171 2618 AD-75162 A-150830 AGAAUGAGGAAUAAUAAGU 5171-5189 2339A-150831 ACUUAUUAUUCCUCAUUCU 5171-5189 2619 AD-75163 A-150832UUAAACCCACAUAGACUCU 5189-5207 2340 A-150833 AGAGUCUAUGUGGGUUUAA5189-5207 2620 AD-75164 A-150834 CUUUAAAACUAUAGGCUAA 5206-5224 2341A-150835 UUAGCCUAUAGUUUUAAAG 5206-5224 2621 AD-75165 A-150836AGAUAGAAAUGUAUGUUUA 5223-5241 2342 A-150837 UAAACAUACAUUUCUAUCU5223-5241 2622 AD-75166 A-150838 UUUGACUUGUUGAAGCUAU 5238-5256 2343A-150839 AUAGCUUCAACAAGUCAAA 5238-5256 2623 AD-75167 A-150840UUUUUAAUCUUAAAAGAUU 5284-5302 2344 A-150841 AAUCUUUUAAGAUUAAAAA5284-5302 2624 AD-75168 A-150842 UUGUGCUAAUUUAUUAGAA 5301-5319 2345A-150843 UUCUAAUAAAUUAGCACAA 5301-5319 2625 AD-75169 A-150844UUAUUAGAGCAGAACCUGU 5311-5329 2346 A-150845 ACAGGUUCUGCUCUAAUAA5311-5329 2626 AD-75170 A-150846 GUUUGGCUCUCCUCAGAAA 5328-5346 2347A-150847 UUUCUGAGGAGAGCCAAAC 5328-5346 2627 AD-75171 A-150848CAAUAUUUUCAAAAGAUAA 5383-5401 2348 A-150849 UUAUCUUUUGAAAAUAUUG5383-5401 2628 AD-75172 A-150850 UCAAAAGAUAAAUCUGAUU 5391-5409 2349A-150851 AAUCAGAUUUAUCUUUUGA 5391-5409 2629 AD-75173 A-150852UUAUGCAAUGGCAUCAUUU 5409-5427 2350 A-150853 AAAUGAUGCCAUUGCAUAA5409-5427 2630 AD-75174 A-150854 UGCAAUGGCAUCAUUUAUU 5412-5430 2351A-150855 AAUAAAUGAUGCCAUUGCA 5412-5430 2631 AD-75175 A-150856UUUAAAACAGAAGAAUUGU 5430-5448 2352 A-150857 ACAAUUCUUCUGUUUUAAA5430-5448 2632 AD-75176 A-150858 AACAACAAAAGGAAAAUGU 5505-5523 2353A-150859 ACAUUUUCCUUUUGUUGUU 5505-5523 2633 AD-75177 A-150860UUAAUCCUGUAGUACAUAU 5570-5588 2354 A-150861 AUAUGUACUACAGGAUUAA5570-5588 2634 AD-75178 A-150862 UUUAAUAUUUUAUAAGACA 5603-5621 2355A-150863 UGUCUUAUAAAAUAUUAAA 5603-5621 2635 AD-75179 A-150864CCUUCCUGUUAGGUAUUAA 5620-5638 2356 A-150865 UUAAUACCUAACAGGAAGG5620-5638 2636 AD-75180 A-150866 UUAGGUAUUAGAAAGUGAU 5628-5646 2357A-150867 AUCACUUUCUAAUACCUAA 5628-5646 2637 AD-75181 A-150868AUACAUAGAUAUCUUUUUU 5645-5663 2358 A-150869 AAAAAAGAUAUCUAUGUAU5645-5663 2638 AD-75182 A-150870 UUUUUGUGUAAUUUCUAUU 5659-5677 2359A-150871 AAUAGAAAUUACACAAAAA 5659-5677 2639 AD-75183 A-150872UUAAAAAAGAGAGAAGACU 5677-5695 2360 A-150873 AGUCUUCUCUCUUUUUUAA5677-5695 2640 AD-75184 A-150874 CUGUCAGAAGCUUUAAGUA 5694-5712 2361A-150875 UACUUAAAGCUUCUGACAG 5694-5712 2641 AD-75185 A-150876UAUGGUACAGGAUAAAGAU 5715-5733 2362 A-150877 AUCUUUAUCCUGUACCAUA5715-5733 2642 AD-75186 A-150878 UUAAAUAACCAAUUCCUAU 5740-5758 2363A-150879 AUAGGAAUUGGUUAUUUAA 5740-5758 2643 AD-75187 A-150880UUGUUUUUUAAAGAAACCU 5773-5791 2364 A-150881 AGGUUUCUUUAAAAAACAA5773-5791 2644 AD-75188 A-150882 CUCUCACAGAUAAGACAGA 5790-5808 2365A-150883 UCUGUCUUAUCUGUGAGAG 5790-5808 2645 AD-75189 A-150884CAGAAUUUUAUAGAGGGCU 5884-5902 2366 A-150885 AGCCCUCUAUAAAAUUCUG5884-5902 2646 AD-75190 A-150886 UCUAGAAUUAAAGGAACCU 5917-5935 2367A-150887 AGGUUCCUUUAAUUCUAGA 5917-5935 2647 AD-75191 A-150888CUCACUGAAAACAUAUAUU 5934-5952 2368 A-150889 AAUAUAUGUUUUCAGUGAG5934-5952 2648 AD-75192 A-150890 AAACAUAUAUUUCACGUGU 5942-5960 2369A-150891 ACACGUGAAAUAUAUGUUU 5942-5960 2649 AD-75193 A-150892GUUCCCUCUUUUUUUUUUU 5959-5977 2370 A-150893 AAAAAAAAAAAGAGGGAAC5959-5977 2650 AD-75194 A-150894 UUAAGCGAUUCUCCUGCCU 6066-6084 2371A-150895 AGGCAGGAGAAUCGCUUAA 6066-6084 2651 AD-75195 A-150896CGGCUAAUUUUUUGGAUUU 6129-6147 2372 A-150897 AAAUCCAAAAAAUUAGCCG6129-6147 2652 AD-75196 A-150898 UUUAAUAGAGACGGGGUUU 6147-6165 2373A-150899 AAACCCCGUCUCUAUUAAA 6147-6165 2653 AD-75197 A-150900UUUACCAUGUUGGCCAGGU 6164-6182 2374 A-150901 ACCUGGCCAACAUGGUAAA6164-6182 2654 AD-75198 A-150902 UUGCUGGGAUUACAGGCAU 6231-6249 2375A-150903 AUGCCUGUAAUCCCAGCAA 6231-6249 2655 AD-75199 A-150904UUAAACAUGAUCCUUCUCU 6303-6321 2376 A-150905 AGAGAAGGAUCAUGUUUAA6303-6321 2656 AD-75200 A-150906 GGGGUCUUUCAAGGGGAAA 6346-6364 2377A-150907 UUUCCCCUUGAAAGACCCC 6346-6364 2657 AD-75201 A-150908AAAAAUCCAAGCUUUUUUA 6364-6382 2378 A-150909 UAAAAAAGCUUGGAUUUUU6364-6382 2658 AD-75202 A-150910 AAAGUAAAAAAAAAAAAAG 6382-6400 2379A-150911 CUUUUUUUUUUUUUACUUU 6382-6400 2659 AD-75203 A-150912AGAGAGGACACAAAACCAA 6399-6417 2380 A-150913 UUGGUUUUGUGUCCUCUCU6399-6417 2660 AD-75204 A-150914 UUAAGAUGGAGACAGAGUU 6444-6462 2381A-150915 AACUCUGUCUCCAUCUUAA 6444-6462 2661 AD-75205 A-150916UUUCUCCUAAUAACCGGAA 6461-6479 2382 A-150917 UUCCGGUUAUUAGGAGAAA6461-6479 2662 AD-75206 A-150918 GCUGAAUUACCUUUCACUU 6479-6497 2383A-150919 AAGUGAAAGGUAAUUCAGC 6479-6497 2663 AD-75207 A-150920UUCAAAAACAUGACCUUCA 6497-6515 2384 A-150921 UGAAGGUCAUGUUUUUGAA6497-6515 2664 AD-75208 A-150922 CAAUCCUUAGAAUCUGCCU 6517-6535 2385A-150923 AGGCAGAUUCUAAGGAUUG 6517-6535 2665 AD-75209 A-150924UUUUAUAUUACUGAGGCCU 6538-6556 2386 A-150925 AGGCCUCAGUAAUAUAAAA6538-6556 2666 AD-75210 A-150926 AAAAGUAAACAUUACUCAU 6557-6575 2387A-150927 AUGAGUAAUGUUUACUUUU 6557-6575 2667 AD-75211 A-150928UUUAUUUUGCCCAAAAUGA 6576-6594 2388 A-150929 UCAUUUUGGGCAAAAUAAA6576-6594 2668 AD-75212 A-150930 CACUGAUGUAAAGUAGGAA 6594-6612 2389A-150931 UUCCUACUUUACAUCAGUG 6594-6612 2669 AD-75213 A-150932AAAAUAAAAACAGAGCUCU 6612-6630 2390 A-150933 AGAGCUCUGUUUUUAUUUU6612-6630 2670 AD-75214 A-150934 CUAAAAUCCCUUUCAAGCA 6629-6647 2391A-150935 UGCUUGAAAGGGAUUUUAG 6629-6647 2671 AD-75215 A-150936UUGACCCCACUCACCAACU 6653-6671 2392 A-150937 AGUUGGUGAGUGGGGUCAA6653-6671 2672 AD-75216 A-150938 UUAUCUUGUACCCGCUGCU 6722-6740 2393A-150939 AGCAGCGGGUACAAGAUAA 6722-6740 2673 AD-75217 A-150940CUGAAACCUCAAGCUGUCU 6762-6780 2394 A-150941 AGACAGCUUGAGGUUUCAG6762-6780 2674 AD-75218 A-150942 GUAUCAUGAAAAUGUCUAU 6806-6824 2395A-150943 AUAGACAUUUUCAUGAUAC 6806-6824 2675 AD-75219 A-150944UUCAAAAUAUCAAAACCUU 6824-6842 2396 A-150945 AAGGUUUUGAUAUUUUGAA6824-6842 2676 AD-75220 A-150946 UUUCAAAUAUCACGCAGCU 6841-6859 2397A-150947 AGCUGCGUGAUAUUUGAAA 6841-6859 2677 AD-75221 A-150948CUUAUAUUCAGUUUACAUA 6858-6876 2398 A-150949 UAUGUAAACUGAAUAUAAG6858-6876 2678 AD-75222 A-150950 UUACAUAAAGGCCCCAAAU 6870-6888 2399A-150951 AUUUGGGGCCUUUAUGUAA 6870-6888 2679 AD-75223 A-150952AUACCAUGUCAGAUCUUUU 6887-6905 2400 A-150953 AAAAGAUCUGACAUGGUAU6887-6905 2680 AD-75224 A-150954 AAAAGAGUUAAUGAACUAU 6910-6928 2401A-150955 AUAGUUCAUUAACUCUUUU 6910-6928 2681 AD-75225 A-150956AUGAGAAUUGGGAUUACAU 6927-6945 2402 A-150957 AUGUAAUCCCAAUUCUCAU6927-6945 2682 AD-75226 A-150958 AUCAUGUAUUUUGCCUCAU 6944-6962 2403A-150959 AUGAGGCAAAAUACAUGAU 6944-6962 2683 AD-75227 A-150960UUAUCACACUUAUAGGCCA 6969-6987 2404 A-150961 UGGCCUAUAAGUGUGAUAA6969-6987 2684 AD-75228 A-150962 CAAGUGUGAUAAAUAAACU 6986-7004 2405A-150963 AGUUUAUUUAUCACACUUG 6986-7004 2685 AD-75229 A-150964UUACAGACACUGAAUUAAU 7004-7022 2406 A-150965 AUUAAUUCAGUGUCUGUAA7004-7022 2686 AD-75230 A-150966 UUUGAAACCAGAAAAUAAU 7035-7053 2407A-150967 AUUAUUUUCUGGUUUCAAA 7035-7053 2687 AD-75231 A-150968AUGACUGGCCAUUCGUUAA 7052-7070 2408 A-150969 UUAACGAAUGGCCAGUCAU7052-7070 2688 AD-75232 A-150970 UUAGUUGAAAAGCAUAUUU 7078-7096 2409A-150971 AAAUAUGCUUUUCAACUAA 7078-7096 2689 AD-75233 A-150972UUUUUAUUAAAUUAAUUCU 7095-7113 2410 A-150973 AGAAUUAAUUUAAUAAAAA7095-7113 2690 AD-75234 A-150974 CUGAUUGUAUUUGAAAUUA 7112-7130 2411A-150975 UAAUUUCAAAUACAAUCAG 7112-7130 2691 AD-75235 A-150976UUUGAAAUUAUUAUUCAAU 7121-7139 2412 A-150977 AUUGAAUAAUAAUUUCAAA7121-7139 2692 AD-75236 A-150978 UUAUGGCAGAGGAAUAUCA 7144-7162 2413A-150979 UGAUAUUCCUCUGCCAUAA 7144-7162 2693 AD-75237 A-150980UCUAAAAAUGUAACUAAUU 7175-7193 2414 A-150981 AAUUAGUUACAUUUUUAGA7175-7193 2694 AD-75238 A-150982 UUUACUGUUUAAUAAGCAU 7210-7228 2415A-150983 AUGCUUAUUAAACAGUAAA 7210-7228 2695 AD-75239 A-150984UGUCAUAAUAAAAUGGUAU 7252-7270 2416 A-150985 AUACCAUUUUAUUAUGACA7252-7270 2696 AD-75240 A-150986 AUAUCUUUCUUUAGUAAUU 7269-7287 2417A-150987 AAUUACUAAAGAAAGAUAU 7269-7287 2697 AD-75241 A-150988UUAGUAAUUACAUUAAAAU 7279-7297 2418 A-150989 AUUUUAAUGUAAUUACUAA7279-7297 2698 AD-75242 A-150990 AUUAGUCAUGUUUGAUUAA 7296-7314 2419A-150991 UUAAUCAAACAUGACUAAU 7296-7314 2699

TABLE 19 IGF-1 in vitro 10 nM screen 10 nM 10 nM Position in Duplex NameAVG STD NM_000618.3 AD-74963 2.44 2.3  6-24 AD-74964 7.2 5.49 24-42AD-74965 3.3 3.72 41-59 AD-74966 4.25 1.91 54-72 AD-74967 15.72 4.372-90 AD-74968 3.11 0.38 127-145 AD-74969 17.28 0.98 185-203 AD-749707.75 1.22 203-221 AD-74971 4.93 4 220-238 AD-74972 21.83 3.83 247-265AD-74973 14.71 5.65 277-295 AD-74974 38.48 1.73 430-448 AD-74975 8.991.93 447-465 AD-74976 22.76 2.57 462-480 AD-74977 34.47 2.7 543-561AD-74978 10.33 10.14 654-672 AD-74979 4.03 0.6 672-690 AD-74980 22.8418.43 750-768 AD-74981 10.09 7.19 774-792 AD-74982 5.27 0.53 792-810AD-74983 7.33 3.35 818-836 AD-74984 25.15 1.05 835-853 AD-74985 9.511.12 852-870 AD-74986 13.08 1.24 894-912 AD-74987 15.81 0.07 912-930AD-74988 74.25 9.8 930-948 AD-74989 53.17 16.23 947-965 AD-74990 34.921.88 1091-1109 AD-74991 35.6 2.75 1108-1126 AD-74992 54.21 4.471125-1143 AD-74993 51.57 3.65 1135-1153 AD-74994 20.06 0.5 1144-1162AD-74995 49.73 0.85 1162-1180 AD-74996 54.67 2.95 1195-1213 AD-7499724.76 9.85 1197-1215 AD-74998 116.31 7.45 1215-1233 AD-74999 37.97 1.631232-1250 AD-75000 17.29 1.27 1293-1311 AD-75001 44.75 3.5 1311-1329AD-75002 33.61 1.4 1334-1352 AD-75003 50.72 3.07 1352-1370 AD-7500449.47 3.37 1370-1388 AD-75005 33.73 0.68 1388-1406 AD-75006 62.64 3.341406-1424 AD-75007 36.36 0.24 1423-1441 AD-75008 37.81 2.85 1440-1458AD-75009 19.35 1.31 1472-1490 AD-75010 71.78 3.37 1494-1512 AD-7501125.82 2.55 1511-1529 AD-75012 55.66 5.16 1528-1546 AD-75013 83.97 4.371572-1590 AD-75014 29.26 11.24 1599-1617 AD-75015 40.19 0.14 1625-1643AD-75016 38.98 4.61 1643-1661 AD-75017 32.96 4.54 1690-1708 AD-7501819.3 1.74 1709-1727 AD-75019 71.36 5.08 1757-1775 AD-75020 20.46 1.941793-1811 AD-75021 33.72 1.42 1807-1825 AD-75022 84.23 3.34 1825-1843AD-75023 24.26 1.03 1843-1861 AD-75024 40.83 2.06 1966-1984 AD-7502548.65 1.28 2016-2034 AD-75026 70.35 5.83 2033-2051 AD-75027 94.74 14.092040-2058 AD-75028 29.93 7.99 2057-2075 AD-75029 31.95 1.57 2090-2108AD-75030 64.72 8.6 2140-2158 AD-75031 44.8 3.93 2170-2188 AD-75032 32.332.6 2192-2210 AD-75033 26.13 0.64 2210-2228 AD-75034 43.7 7.02 2228-2246AD-75035 70.89 7.82 2249-2267 AD-75036 106.4 4.66 2266-2284 AD-7503747.27 4.85 2285-2303 AD-75038 48.86 4.15 2303-2321 AD-75039 40.63 4.832322-2340 AD-75040 107.1 7.22 2340-2358 AD-75041 35.81 5.21 2357-2375AD-75042 52.59 6.79 2398-2416 AD-75043 55.9 9.3 2432-2450 AD-75044 46.6611.14 2449-2467 AD-75045 82.13 13.7 2453-2471 AD-75046 73.55 5.152470-2488 AD-75047 71.67 15.96 2482-2500 AD-75048 38.18 30.65 2521-2539AD-75049 92.97 13.14 2539-2557 AD-75050 86 15.13 2548-2566 AD-7505159.23 8.69 2566-2584 AD-75052 104.39 19.17 2584-2602 AD-75053 58.6610.73 2601-2619 AD-75054 89.3 16.07 2618-2636 AD-75055 64.45 10.532623-2641 AD-75056 95.7 23.97 2640-2658 AD-75057 38.59 5.32 2648-2666AD-75058 35.56 5.16 2666-2684 AD-75059 62.95 9.3 2684-2702 AD-7506050.41 11.52 2771-2789 AD-75061 34.92 9.92 2793-2811 AD-75062 52.01 10.492811-2829 AD-75063 49.98 9.93 2828-2846 AD-75064 113.1 26.07 2869-2887AD-75065 73.65 7.55 2906-2924 AD-75066 43.56 5.6 2920-2938 AD-7506756.32 9.44 2938-2956 AD-75068 57.61 8.64 2956-2974 AD-75069 34.69 12.052962-2980 AD-75070 89.29 14.15 2980-2998 AD-75071 64.02 16.69 3006-3024AD-75072 125.6 49.27 3025-3043 AD-75073 111.64 19.51 3043-3061 AD-7507475.61 13.18 3060-3078 AD-75075 111.51 16.74 3077-3095 AD-75076 69.43 9.43100-3118 AD-75077 44.04 5 3137-3155 AD-75078 57.27 10.2 3155-3173AD-75079 28.28 5.64 3208-3226 AD-75080 59.53 7.5 3225-3243 AD-7508161.41 5.87 3234-3252 AD-75082 54.31 7.14 3252-3270 AD-75083 34.99 5.513269-3287 AD-75084 46.86 9.9 3277-3295 AD-75085 56.82 7.96 3294-3312AD-75086 58.83 11.06 3334-3352 AD-75087 55.26 2.93 3352-3370 AD-7508834.12 16.28 3369-3387 AD-75089 63.74 11.09 3400-3418 AD-75090 36.81 7.733417-3435 AD-75091 31.56 5.56 3449-3467 AD-75092 61.39 11.47 3517-3535AD-75093 83.2 19.37 3535-3553 AD-75094 80.16 13.64 3567-3585 AD-7509538.33 8.26 3585-3603 AD-75096 103.57 22.84 3603-3621 AD-75097 69.98 7.033621-3639 AD-75098 51.57 14.6 3662-3680 AD-75099 31.97 13.18 3686-3704AD-75100 94.94 9.26 3704-3722 AD-75101 36.43 11.54 3721-3739 AD-7510270.66 7.75 3732-3750 AD-75103 57.38 5.65 3750-3768 AD-75104 77.58 15.393767-3785 AD-75105 70.13 8.3 3770-3788 AD-75106 50.24 6.25 3788-3806AD-75107 34.8 6.73 3806-3824 AD-75108 58.82 3.73 3835-3853 AD-7510965.08 10.73 3865-3883 AD-75110 31.63 14.97 3939-3957 AD-75111 5.82 0.913982-4000 AD-75112 11.18 0.76 4000-4018 AD-75113 38.66 8.55 4081-4099AD-75114 14.58 5.96 4098-4116 AD-75115 12.98 2.49 4101-4119 AD-7511635.3 6.1 4119-4137 AD-75117 57.1 9.56 4137-4155 AD-75118 23.23 4.434154-4172 AD-75119 54.12 7.09 4208-4226 AD-75120 15.15 2.22 4271-4289AD-75121 19.41 4.81 4289-4307 AD-75122 33.51 8.79 4319-4337 AD-7512310.61 1.98 4336-4354 AD-75124 22.01 6.31 4341-4359 AD-75125 10.88 0.884359-4377 AD-75126 84.91 10.8 4377-4395 AD-75127 71.33 14.6 4395-4413AD-75128 78.44 5.21 4412-4430 AD-75129 76.77 25.96 4426-4444 AD-7513015.56 6.25 4443-4461 AD-75131 13.16 4.18 4460-4478 AD-75132 86.74 17.914464-4482 AD-75133 36.15 4.26 4484-4502 AD-75134 51.96 9.57 4511-4529AD-75135 19.14 4.52 4566-4584 AD-75136 43.64 6.37 4584-4602 AD-75137 3.80.43 4601-4619 AD-75138 40.31 6.39 4625-4643 AD-75139 20.1 4.154642-4660 AD-75140 53.32 1.96 4645-4663 AD-75141 51.87 3.72 4669-4687AD-75142 32.17 4.32 4686-4704 AD-75143 17.5 6.52 4699-4717 AD-7514494.26 24.89 4717-4735 AD-75145 8.73 1.94 4734-4752 AD-75146 39.8 9.224751-4769 AD-75147 44.81 9.36 4770-4788 AD-75148 21.99 3.67 4780-4798AD-75149 56.19 8.99 4798-4816 AD-75150 22.7 2.67 4815-4833 AD-7515133.03 2.82 4825-4843 AD-75152 28.29 11.66 4884-4902 AD-75153 34.14 6.634911-4929 AD-75154 43.03 6.81 4987-5005 AD-75155 10.11 2.68 5004-5022AD-75156 49.77 4.33 5016-5034 AD-75157 10.55 1.54 5050-5068 AD-7515898.46 15.78 5098-5116 AD-75159 59.88 7.14 5115-5133 AD-75160 67.78 10.535127-5145 AD-75161 71.58 12.59 5153-5171 AD-75162 9.5 2.65 5171-5189AD-75163 99.59 10.77 5189-5207 AD-75164 24.32 6.23 5206-5224 AD-7516548.65 10.6 5223-5241 AD-75166 20.31 3.83 5238-5256 AD-75167 96.76 8.315284-5302 AD-75168 50.33 6.42 5301-5319 AD-75169 35.41 3.61 5311-5329AD-75170 9.16 1.79 5328-5346 AD-75171 74.55 6.92 5383-5401 AD-7517261.38 10.44 5391-5409 AD-75173 18.61 2.81 5409-5427 AD-75174 12.46 3.745412-5430 AD-75175 64.04 8.99 5430-5448 AD-75176 47.5 11.54 5505-5523AD-75177 26.63 1.26 5570-5588 AD-75178 93.5 13.15 5603-5621 AD-7517913.82 1.72 5620-5638 AD-75180 40.37 3.65 5628-5646 AD-75181 68.1 145645-5663 AD-75182 91.45 7.76 5659-5677 AD-75183 52.7 9.56 5677-5695AD-75184 10.64 1.91 5694-5712 AD-75185 22.43 3.75 5715-5733 AD-7518639.19 4.23 5740-5758 AD-75187 80.34 23.05 5773-5791 AD-75188 18.72 4.875790-5808 AD-75189 71.77 15.33 5884-5902 AD-75190 69.3 7.75 5917-5935AD-75191 13.99 6.28 5934-5952 AD-75192 83.17 9.31 5942-5960 AD-7519379.66 18.37 5959-5977 AD-75194 64.7 6.93 6066-6084 AD-75195 79.21 5.636129-6147 AD-75196 105.5 9.43 6147-6165 AD-75197 128.21 12.85 6164-6182AD-75198 46.08 7.51 6231-6249 AD-75199 117.2 7.51 6303-6321 AD-7520050.47 12.5 6346-6364 AD-75201 59.91 14.09 6364-6382 AD-75202 107.5322.57 6382-6400 AD-75203 25.97 4.96 6399-6417 AD-75204 71.96 3.586444-6462 AD-75205 39.19 13.83 6461-6479 AD-75206 11.68 4.11 6479-6497AD-75207 40.79 10.66 6497-6515 AD-75208 34.43 5.15 6517-6535 AD-75209107.98 25.75 6538-6556 AD-75210 52.4 6.95 6557-6575 AD-75211 90.01 19.176576-6594 AD-75212 22.91 6.75 6594-6612 AD-75213 87.12 9.51 6612-6630AD-75214 19.83 5.95 6629-6647 AD-75215 50.88 6.82 6653-6671 AD-7521643.88 4.7 6722-6740 AD-75217 18.19 2.83 6762-6780 AD-75218 9.91 1.36806-6824 AD-75219 54.72 5.78 6824-6842 AD-75220 60.73 10.07 6841-6859AD-75221 23.15 1.19 6858-6876 AD-75222 36.29 4.92 6870-6888 AD-7522321.88 2.24 6887-6905 AD-75224 32.13 3.75 6910-6928 AD-75225 12.65 3.496927-6945 AD-75226 48.19 14.5 6944-6962 AD-75227 58.51 8.21 6969-6987AD-75228 53.16 6.09 6986-7004 AD-75229 10.94 2.92 7004-7022 AD-7523031.83 5.18 7035-7053 AD-75231 15.75 2.98 7052-7070 AD-75232 18.71 3.477078-7096 AD-75233 106.56 8.46 7095-7113 AD-75234 18.49 4.54 7112-7130AD-75235 113.68 22.34 7121-7139 AD-75236 20.92 10.52 7144-7162 AD-7523729.1 4.1 7175-7193 AD-75238 39.59 10.16 7210-7228 AD-75239 23.08 3.817252-7270 AD-75240 98.59 16.79 7269-7287 AD-75241 109.49 8.89 7279-7297AD-75242 92.6 7.78 7296-7314

TABLE 20 Modified Sense and Antisense Strand Sequences of IGF-1 dsRNAsSense SEQ Antisense SEQ SEQ Duplex Oligo ID Oligo ID ID Name NameSense sequence NO Name Antisense sequence NO mRNA target sequence NOAD-74963 A-150432 UAGAUAAAUGUGAGGAUUUdTdT 2700 A-150433AAAUCCUCACAUUUAUCUAdTdT 2980 UAGAUAAAUGUGAGGAUUU 3260 AD-74964 A-150434UUCUCUAAAUCCCUCUUCUdTdT 2701 A-150435 AGAAGAGGGAUUUAGAGAAdTdT 2981UUCUCUAAAUCCCUCUUCU 3261 AD-74965 A-150436 CUGUUUGCUAAAUCUCACUdTdT 2702A-150437 AGUGAGAUUUAGCAAACAGdTdT 2982 CUGUUUGCUAAAUCUCACU 3262 AD-74966A-150438 CUCACUGUCACUGCUAAAUdTdT 2703 A-150439 AUUUAGCAGUGACAGUGAGdTdT2983 CUCACUGUCACUGCUAAAU 3263 AD-74967 A-150440 UUCAGAGCAGAUAGAGCCUdTdT2704 A-150441 AGGCUCUAUCUGCUCUGAAdTdT 2984 UUCAGAGCAGAUAGAGCCU 3264AD-74968 A-150442 CAUUGCUCUCAACAUCUCAdTdT 2705 A-150443UGAGAUGUUGAGAGCAAUGdTdT 2985 CAUUGCUCUCAACAUCUCC 3265 AD-74969 A-150444ACCAAUUCAUUUUCAGACUdTdT 2706 A-150445 AGUCUGAAAAUGAAUUGGUdTdT 2986ACCAAUUCAUUUUCAGACU 3266 AD-74970 A-150446 UUUGUACUUCAGAAGCAAUdTdT 2707A-150447 AUUGCUUCUGAAGUACAAAdTdT 2987 UUUGUACUUCAGAAGCAAU 3267 AD-74971A-150448 AUGGGAAAAAUCAGCAGUAdTdT 2708 A-150449 UACUGCUGAUUUUUCCCAUdTdT2988 AUGGGAAAAAUCAGCAGUC 3268 AD-74972 A-150450 CAAUUAUUUAAGUGCUGCUdTdT2709 A-150451 AGCAGCACUUAAAUAAUUGdTdT 2989 CAAUUAUUUAAGUGCUGCU 3269AD-74973 A-150452 UUGAAGGUGAAGAUGCACAdTdT 2710 A-150453UGUGCAUCUUCACCUUCAAdTdT 2990 UUGAAGGUGAAGAUGCACA 3270 AD-74974 A-150454UUUUAUUUCAACAAGCCCAdTdT 2711 A-150455 UGGGCUUGUUGAAAUAAAAdTdT 2991UUUUAUUUCAACAAGCCCA 3271 AD-74975 A-150456 CACAGGGUAUGGCUCCAGAdTdT 2712A-150457 UCUGGAGCCAUACCCUGUGdTdT 2992 CACAGGGUAUGGCUCCAGC 3272 AD-74976A-150458 CAGCAGUCGGAGGGCGCCUdTdT 2713 A-150459 AGGCGCCCUCCGACUGCUGdTdT2993 CAGCAGUCGGAGGGCGCCU 3273 AD-74977 A-150460 UUGCGCACCCCUCAAGCCUdTdT2714 A-150461 AGGCUUGAGGGGUGCGCAAdTdT 2994 UUGCGCACCCCUCAAGCCU 3274AD-74978 A-150462 UGCAGGAAACAAGAACUAAdTdT 2715 A-150463UUAGUUCUUGUUUCCUGCAdTdT 2995 UGCAGGAAACAAGAACUAC 3275 AD-74979 A-150464CAGGAUGUAGGAAGACCCUdTdT 2716 A-150465 AGGGUCUUCCUACAUCCUGdTdT 2996CAGGAUGUAGGAAGACCCU 3276 AD-74980 A-150466 UUAAACUUUGGAACACCUAdTdT 2717A-150467 UAGGUGUUCCAAAGUUUAAdTdT 2997 UUAAACUUUGGAACACCUA 3277 AD-74981A-150468 AAAUAAGUUUGAUAACAUUdTdT 2718 A-150469 AAUGUUAUCAAACUUAUUUdTdT2998 AAAUAAGUUUGAUAACAUU 3278 AD-74982 A-150470 UUAAAAGAUGGGCGUUUCAdTdT2719 A-150471 UGAAACGCCCAUCUUUUAAdTdT 2999 UUAAAAGAUGGGCGUUUCC 3279AD-74983 A-150472 AAAUACACAAGUAAACAUUdTdT 2720 A-150473AAUGUUUACUUGUGUAUUUdTdT 3000 AAAUACACAAGUAAACAUU 3280 AD-74984 A-150474UUCCAACAUUGUCUUUAGAdTdT 2721 A-150475 UCUAAAGACAAUGUUGGAAdTdT 3001UUCCAACAUUGUCUUUAGG 3281 AD-74985 A-150476 GGAGUGAUUUGCACCUUGAdTdT 2722A-150477 UCAAGGUGCAAAUCACUCCdTdT 3002 GGAGUGAUUUGCACCUUGC 3282 AD-74986A-150478 AUUGCUGUUGAUCUUUUAUdTdT 2723 A-150479 AUAAAAGAUCAACAGCAAUdTdT3003 AUUGCUGUUGAUCUUUUAU 3283 AD-74987 A-150480 UCAAUAAUGUUCUAUAGAAdTdT2724 A-150481 UUCUAUAGAACAUUAUUGAdTdT 3004 UCAAUAAUGUUCUAUAGAA 3284AD-74988 A-150482 AAAGAAAAAAAAAAUAUAUdTdT 2725 A-150483AUAUAUUUUUUUUUUCUUUdTdT 3005 AAAGAAAAAAAAAAUAUAU 3285 AD-74989 A-150484AUAUAUAUAUAUAUCUUAAdTdT 2726 A-150485 UUAAGAUAUAUAUAUAUAUdTdT 3006AUAUAUAUAUAUAUCUUAG 3286 AD-74990 A-150486 UUUCCUUAUUUGCACUUCUdTdT 2727A-150487 AGAAGUGCAAAUAAGGAAAdTdT 3007 UUUCCUUAUUUGCACUUCU 3287 AD-74991A-150488 CUUUCUACACAACUCGGGAdTdT 2728 A-150489 UCCCGAGUUGUGUAGAAAGdTdT3008 CUUUCUACACAACUCGGGC 3288 AD-74992 A-150490 GCUGUUUGUUUUACAGUGUdTdT2729 A-150491 ACACUGUAAAACAAACAGCdTdT 3009 GCUGUUUGUUUUACAGUGU 3289AD-74993 A-150492 UUACAGUGUCUGAUAAUCUdTdT 2730 A-150493AGAUUAUCAGACACUGUAAdTdT 3010 UUACAGUGUCUGAUAAUCU 3290 AD-74994 A-150494CUGAUAAUCUUGUUAGUCUdTdT 2731 A-150495 AGACUAACAAGAUUAUCAGdTdT 3011CUGAUAAUCUUGUUAGUCU 3291 AD-74995 A-150496 UAUACCCACCACCUCCCUUdTdT 2732A-150497 AAGGGAGGUGGUGGGUAUAdTdT 3012 UAUACCCACCACCUCCCUU 3292 AD-74996A-150498 UUGCCGAAUUUGGCCUCCUdTdT 2733 A-150499 AGGAGGCCAAAUUCGGCAAdTdT3013 UUGCCGAAUUUGGCCUCCU 3293 AD-74997 A-150500 GCCGAAUUUGGCCUCCUCAdTdT2734 A-150501 UGAGGAGGCCAAAUUCGGCdTdT 3014 GCCGAAUUUGGCCUCCUCA 3294AD-74998 A-150502 AAAAGCAGCAGCAAGUCGUdTdT 2735 A-150503ACGACUUGCUGCUGCUUUUdTdT 3015 AAAAGCAGCAGCAAGUCGU 3295 AD-74999 A-150504GUCAAGAAGCACACCAAUUdTdT 2736 A-150505 AAUUGGUGUGCUUCUUGACdTdT 3016GUCAAGAAGCACACCAAUU 3296 AD-75000 A-150506 AGUUGGAUGCAUUUUAUUUdTdT 2737A-150507 AAAUAAAAUGCAUCCAACUdTdT 3017 AGUUGGAUGCAUUUUAUUU 3297 AD-75001A-150508 UUAGACACAAAGCUUUAUUdTdT 2738 A-150509 AAUAAAGCUUUGUGUCUAAdTdT3018 UUAGACACAAAGCUUUAUU 3298 AD-75002 A-150510 CACAUCAUGCUUACAAAAAdTdT2739 A-150511 UUUUUGUAAGCAUGAUGUGdTdT 3019 CACAUCAUGCUUACAAAAA 3299AD-75003 A-150512 AAGAAUAAUGCAAAUAGUUdTdT 2740 A-150513AACUAUUUGCAUUAUUCUUdTdT 3020 AAGAAUAAUGCAAAUAGUU 3300 AD-75004 A-150514UGCAACUUUGAGGCCAAUAdTdT 2741 A-150515 UAUUGGCCUCAAAGUUGCAdTdT 3021UGCAACUUUGAGGCCAAUC 3301 AD-75005 A-150516 CAUUUUUAGGCAUAUGUUUdTdT 2742A-150517 AAACAUAUGCCUAAAAAUGdTdT 3022 CAUUUUUAGGCAUAUGUUU 3302 AD-75006A-150518 UUAAACAUAGAAAGUUUCUdTdT 2743 A-150519 AGAAACUUUCUAUGUUUAAdTdT3023 UUAAACAUAGAAAGUUUCU 3303 AD-75007 A-150520 CUUCAACUCAAAAGAGUUAdTdT2744 A-150521 UAACUCUUUUGAGUUGAAGdTdT 3024 CUUCAACUCAAAAGAGUUC 3304AD-75008 A-150522 UCCUUCAAAUGAUGAGUUAdTdT 2745 A-150523UAACUCAUCAUUUGAAGGAdTdT 3025 UCCUUCAAAUGAUGAGUUA 3305 AD-75009 A-150524UUAGUAACUUUCCUCUUUUdTdT 2746 A-150525 AAAAGAGGAAAGUUACUAAdTdT 3026UUAGUAACUUUCCUCUUUU 3306 AD-75010 A-150526 UUUUUCCAUAUAGAGCACUdTdT 2747A-150527 AGUGCUCUAUAUGGAAAAAdTdT 3027 UUUUUCCAUAUAGAGCACU 3307 AD-75011A-150528 CUAUGUAAAUUUAGCAUAUdTdT 2748 A-150529 AUAUGCUAAAUUUACAUAGdTdT3028 CUAUGUAAAUUUAGCAUAU 3308 AD-75012 A-150530 AUCAAUUAUACAGGAUAUAdTdT2749 A-150531 UAUAUCCUGUAUAAUUGAUdTdT 3029 AUCAAUUAUACAGGAUAUA 3309AD-75013 A-150532 UUUAGUAUAAUGGUGCUAUdTdT 2750 A-150533AUAGCACCAUUAUACUAAAdTdT 3030 UUUAGUAUAAUGGUGCUAU 3310 AD-75014 A-150534UUGUUAUAUGAAAGAGUCUdTdT 2751 A-150535 AGACUCUUUCAUAUAACAAdTdT 3031UUGUUAUAUGAAAGAGUCU 3311 AD-75015 A-150536 ACGGUAAUACGUGAAAGCAdTdT 2752A-150537 UGCUUUCACGUAUUACCGUdTdT 3032 ACGGUAAUACGUGAAAGCA 3312 AD-75016A-150538 AAAACAAUAGGGGAAGCCUdTdT 2753 A-150539 AGGCUUCCCCUAUUGUUUUdTdT3033 AAAACAAUAGGGGAAGCCU 3313 AD-75017 A-150540 UACUGAAAACACCAUCCAUdTdT2754 A-150541 AUGGAUGGUGUUUUCAGUAdTdT 3034 UACUGAAAACACCAUCCAU 3314AD-75018 A-150542 UUGGGAAAGAAGGCAAAGUdTdT 2755 A-150543ACUUUGCCUUCUUUCCCAAdTdT 3035 UUGGGAAAGAAGGCAAAGU 3315 AD-75019 A-150544UCAGACACAAAAGUCCACUdTdT 2756 A-150545 AGUGGACUUUUGUGUCUGAdTdT 3036UCAGACACAAAAGUCCACU 3316 AD-75020 A-150546 CGAGUCCAGAGAGGAAACUdTdT 2757A-150547 AGUUUCCUCUCUGGACUCGdTdT 3037 CGAGUCCAGAGAGGAAACU 3317 AD-75021A-150548 AAACUGUGGAAUGGAAAAAdTdT 2758 A-150549 UUUUUCCAUUCCACAGUUUdTdT3038 AAACUGUGGAAUGGAAAAA 3318 AD-75022 A-150550 AGCAGAAGGCUAGGAAUUUdTdT2759 A-150551 AAAUUCCUAGCCUUCUGCUdTdT 3039 AGCAGAAGGCUAGGAAUUU 3319AD-75023 A-150552 UUAGCAGUCCUGGUUUCUUdTdT 2760 A-150553AAGAAACCAGGACUGCUAAdTdT 3040 UUAGCAGUCCUGGUUUCUU 3320 AD-75024 A-150554CAAAAUGGGGGCAAUAUGUdTdT 2761 A-150555 ACAUAUUGCCCCCAUUUUGdTdT 3041CAAAAUGGGGGCAAUAUGU 3321 AD-75025 A-150556 UUUAAAAAGAUAAAGAUUAdTdT 2762A-150557 UAAUCUUUAUCUUUUUAAAdTdT 3042 UUUAAAAAGAUAAAGAUUC 3322 AD-75026A-150558 UCAGAUUUUUUUUACCCUAdTdT 2763 A-150559 UAGGGUAAAAAAAAUCUGAdTdT3043 UCAGAUUUUUUUUACCCUG 3323 AD-75027 A-150560 UUUUUUACCCUGGGUUGCUdTdT2764 A-150561 AGCAACCCAGGGUAAAAAAdTdT 3044 UUUUUUACCCUGGGUUGCU 3324AD-75028 A-150562 CUGUAAGGGUGCAACAUCAdTdT 2765 A-150563UGAUGUUGCACCCUUACAGdTdT 3045 CUGUAAGGGUGCAACAUCA 3325 AD-75029 A-150564CUGAGAUGCAAGGAAUUCUdTdT 2766 A-150565 AGAAUUCCUUGCAUCUCAGdTdT 3046CUGAGAUGCAAGGAAUUCU 3326 AD-75030 A-150566 UUGGUGAAUUGAAUGCUCAdTdT 2767A-150567 UGAGCAUUCAAUUCACCAAdTdT 3047 UUGGUGAAUUGAAUGCUCC 3327 AD-75031A-150568 UUCUUGUCAGUGAAGCUAUdTdT 2768 A-150569 AUAGCUUCACUGACAAGAAdTdT3048 UUCUUGUCAGUGAAGCUAU 3328 AD-75032 A-150570 AAUAACUGGCCAACUAGUUdTdT2769 A-150571 AACUAGUUGGCCAGUUAUUdTdT 3049 AAUAACUGGCCAACUAGUU 3329AD-75033 A-150572 UGUUAAAAGCUAACAGCUAdTdT 2770 A-150573UAGCUGUUAGCUUUUAACAdTdT 3050 UGUUAAAAGCUAACAGCUC 3330 AD-75034 A-150574CAAUCUCUUAAAACACUUUdTdT 2771 A-150575 AAAGUGUUUUAAGAGAUUGdTdT 3051CAAUCUCUUAAAACACUUU 3331 AD-75035 A-150576 AAAAUAUGUGGGAAGCAUUdTdT 2772A-150577 AAUGCUUCCCACAUAUUUUdTdT 3052 AAAAUAUGUGGGAAGCAUU 3332 AD-75036A-150578 UUUGAUUUUCAAUUUGAUUdTdT 2773 A-150579 AAUCAAAUUGAAAAUCAAAdTdT3053 UUUGAUUUUCAAUUUGAUU 3333 AD-75037 A-150580 UUGAAUUCUGCAUUUGGUUdTdT2774 A-150581 AACCAAAUGCAGAAUUCAAdTdT 3054 UUGAAUUCUGCAUUUGGUU 3334AD-75038 A-150582 UUUAUGAAUACAAAGAUAAdTdT 2775 A-150583UUAUCUUUGUAUUCAUAAAdTdT 3055 UUUAUGAAUACAAAGAUAA 3335 AD-75039 A-150584GUGAAAAGAGAGAAAGGAAdTdT 2776 A-150585 UUCCUUUCUCUCUUUUCACdTdT 3056GUGAAAAGAGAGAAAGGAA 3336 AD-75040 A-150586 AAAGAAAAAGGAGAAAAACdTdT 2777A-150587 GUUUUUCUCCUUUUUCUUUdTdT 3057 AAAGAAAAAGGAGAAAAAC 3337 AD-75041A-150588 ACAAAGAGAUUUCUACCAAdTdT 2778 A-150589 UUGGUAGAAAUCUCUUUGUdTdT3058 ACAAAGAGAUUUCUACCAG 3338 AD-75042 A-150590 UUGUUAGCACUCACUGACUdTdT2779 A-150591 AGUCAGUGAGUGCUAACAAdTdT 3059 UUGUUAGCACUCACUGACU 3339AD-75043 A-150592 UACAUAUCUAGUAAAACCUdTdT 2780 A-150593AGGUUUUACUAGAUAUGUAdTdT 3060 UACAUAUCUAGUAAAACCU 3340 AD-75044 A-150594CUCGUUUAAUACUAUAAAUdTdT 2781 A-150595 AUUUAUAGUAUUAAACGAGdTdT 3061CUCGUUUAAUACUAUAAAU 3341 AD-75045 A-150596 UUUAAUACUAUAAAUAAUAdTdT 2782A-150597 UAUUAUUUAUAGUAUUAAAdTdT 3062 UUUAAUACUAUAAAUAAUA 3342 AD-75046A-150598 UAUUCUAUUCAUUUUGAAAdTdT 2783 A-150599 UUUCAAAAUGAAUAGAAUAdTdT3063 UAUUCUAUUCAUUUUGAAA 3343 AD-75047 A-150600 UUUGAAAAACACAAUGAUUdTdT2784 A-150601 AAUCAUUGUGUUUUUCAAAdTdT 3064 UUUGAAAAACACAAUGAUU 3344AD-75048 A-150602 AAGGAAAGUGAUCCAAAAUdTdT 2785 A-150603AUUUUGGAUCACUUUCCUUdTdT 3065 AAGGAAAGUGAUCCAAAAU 3345 AD-75049 A-150604UUUGAAAUAUUAAAAUAAUdTdT 2786 A-150605 AUUAUUUUAAUAUUUCAAAdTdT 3066UUUGAAAUAUUAAAAUAAU 3346 AD-75050 A-150606 UUAAAAUAAUAUCUAAUAAdTdT 2787A-150607 UUAUUAGAUAUUAUUUUAAdTdT 3067 UUAAAAUAAUAUCUAAUAA 3347 AD-75051A-150608 AAAAGUCACAAAGUUAUCUdTdT 2788 A-150609 AGAUAACUUUGUGACUUUUdTdT3068 AAAAGUCACAAAGUUAUCU 3348 AD-75052 A-150610 UUCUUUAACAAACUUUACUdTdT2789 A-150611 AGUAAAGUUUGUUAAAGAAdTdT 3069 UUCUUUAACAAACUUUACU 3349AD-75053 A-150612 CUCUUAUUCUUAGCUGUAUdTdT 2790 A-150613AUACAGCUAAGAAUAAGAGdTdT 3070 CUCUUAUUCUUAGCUGUAU 3350 AD-75054 A-150614AUAUACAUUUUUUUAAAAGdTdT 2791 A-150615 CUUUUAAAAAAAUGUAUAUdTdT 3071AUAUACAUUUUUUUAAAAG 3351 AD-75055 A-150616 CAUUUUUUUAAAAGUUUGUdTdT 2792A-150617 ACAAACUUUUAAAAAAAUGdTdT 3072 CAUUUUUUUAAAAGUUUGU 3352 AD-75056A-150618 GUUAAAAUAUGCUUGACUAdTdT 2793 A-150619 UAGUCAAGCAUAUUUUAACdTdT3073 GUUAAAAUAUGCUUGACUA 3353 AD-75057 A-150620 AUGCUUGACUAGAGUUUCAdTdT2794 A-150621 UGAAACUCUAGUCAAGCAUdTdT 3074 AUGCUUGACUAGAGUUUCC 3354AD-75058 A-150622 CAGUUGAAAGGCAAAAACUdTdT 2795 A-150623AGUUUUUGCCUUUCAACUGdTdT 3075 CAGUUGAAAGGCAAAAACU 3355 AD-75059 A-150624UUCCAUCACAACAAGAAAUdTdT 2796 A-150625 AUUUCUUGUUGUGAUGGAAdTdT 3076UUCCAUCACAACAAGAAAU 3356 AD-75060 A-150626 UUGGUAUCAAGAAAGUCCAdTdT 2797A-150627 UGGACUUUCUUGAUACCAAdTdT 3077 UUGGUAUCAAGAAAGUCCA 3357 AD-75061A-150628 GUUAGUGUACUAGUCCAUAdTdT 2798 A-150629 UAUGGACUAGUACACUAACdTdT3078 GUUAGUGUACUAGUCCAUC 3358 AD-75062 A-150630 CAUAGCCUAGAAAAUGAUAdTdT2799 A-150631 UAUCAUUUUCUAGGCUAUGdTdT 3079 CAUAGCCUAGAAAAUGAUC 3359AD-75063 A-150632 UCCCUAUCUGCAGAUCAAAdTdT 2800 A-150633UUUGAUCUGCAGAUAGGGAdTdT 3080 UCCCUAUCUGCAGAUCAAG 3360 AD-75064 A-150634UUAUCCAGCAUUCAGAUCUdTdT 2801 A-150635 AGAUCUGAAUGCUGGAUAAdTdT 3081UUAUCCAGCAUUCAGAUCU 3361 AD-75065 A-150636 UUUUUGGUUAAAAGUACCAdTdT 2802A-150637 UGGUACUUUUAACCAAAAAdTdT 3082 UUUUUGGUUAAAAGUACCC 3362 AD-75066A-150638 UACCCAGGCUUGAUUAUUUdTdT 2803 A-150639 AAAUAAUCAAGCCUGGGUAdTdT3083 UACCCAGGCUUGAUUAUUU 3363 AD-75067 A-150640 UCAUGCAAAUUCUAUAUUUdTdT2804 A-150641 AAAUAUAGAAUUUGCAUGAdTdT 3084 UCAUGCAAAUUCUAUAUUU 3364AD-75068 A-150642 UUACAUUCUUGGAAAGUCUdTdT 2805 A-150643AGACUUUCCAAGAAUGUAAdTdT 3085 UUACAUUCUUGGAAAGUCU 3365 AD-75069 A-150644UCUUGGAAAGUCUAUAUGAdTdT 2806 A-150645 UCAUAUAGACUUUCCAAGAdTdT 3086UCUUGGAAAGUCUAUAUGA 3366 AD-75070 A-150646 AAAAACAAAAAUAACAUCUdTdT 2807A-150647 AGAUGUUAUUUUUGUUUUUdTdT 3087 AAAAACAAAAAUAACAUCU 3367 AD-75071A-150648 UUCUCCCACUGGGUCACCUdTdT 2808 A-150649 AGGUGACCCAGUGGGAGAAdTdT3088 UUCUCCCACUGGGUCACCU 3368 AD-75072 A-150650 CAAGGAUCAGAGGCCAGGAdTdT2809 A-150651 UCCUGGCCUCUGAUCCUUGdTdT 3089 CAAGGAUCAGAGGCCAGGA 3369AD-75073 A-150652 AAAAAAAAAAAAAAGACUAdTdT 2810 A-150653UAGUCUUUUUUUUUUUUUUdTdT 3090 AAAAAAAAAAAAAAGACUC 3370 AD-75074 A-150654UCCCUGGAUCUCUGAAUAUdTdT 2811 A-150655 AUAUUCAGAGAUCCAGGGAdTdT 3091UCCCUGGAUCUCUGAAUAU 3371 AD-75075 A-150656 AUAUGCAAAAAGAAGGCCAdTdT 2812A-150657 UGGCCUUCUUUUUGCAUAUdTdT 3092 AUAUGCAAAAAGAAGGCCC 3372 AD-75076A-150658 UAGUGGAGCCAGCAAUCCUdTdT 2813 A-150659 AGGAUUGCUGGCUCCACUAdTdT3093 UAGUGGAGCCAGCAAUCCU 3373 AD-75077 A-150660 UUAACUCUCAGUCCAACAUdTdT2814 A-150661 AUGUUGGACUGAGAGUUAAdTdT 3094 UUAACUCUCAGUCCAACAU 3374AD-75078 A-150662 UUAUUUGAAUUGAGCACCUdTdT 2815 A-150663AGGUGCUCAAUUCAAAUAAdTdT 3095 UUAUUUGAAUUGAGCACCU 3375 AD-75079 A-150664CAGAUGUAAAAGAAACUAUdTdT 2816 A-150665 AUAGUUUCUUUUACAUCUGdTdT 3096CAGAUGUAAAAGAAACUAU 3376 AD-75080 A-150666 AUACAUCAUUUUUGCCCUAdTdT 2817A-150667 UAGGGCAAAAAUGAUGUAUdTdT 3097 AUACAUCAUUUUUGCCCUC 3377 AD-75081A-150668 UUUUGCCCUCUGCCUGUUUdTdT 2818 A-150669 AAACAGGCAGAGGGCAAAAdTdT3098 UUUUGCCCUCUGCCUGUUU 3378 AD-75082 A-150670 UUCCAGACAUACAGGUUCUdTdT2819 A-150671 AGAACCUGUAUGUCUGGAAdTdT 3099 UUCCAGACAUACAGGUUCU 3379AD-75083 A-150672 CUGUGGAAUAAGAUACUGAdTdT 2820 A-150673UCAGUAUCUUAUUCCACAGdTdT 3100 CUGUGGAAUAAGAUACUGG 3380 AD-75084 A-150674UAAGAUACUGGACUCCUCUdTdT 2821 A-150675 AGAGGAGUCCAGUAUCUUAdTdT 3101UAAGAUACUGGACUCCUCU 3381 AD-75085 A-150676 CUUCCCAAGAUGGCACUUAdTdT 2822A-150677 UAAGUGCCAUCUUGGGAAGdTdT 3102 CUUCCCAAGAUGGCACUUC 3382 AD-75086A-150678 GUGUACCUUUUAAAAUUAUdTdT 2823 A-150679 AUAAUUUUAAAAGGUACACdTdT3103 GUGUACCUUUUAAAAUUAU 3383 AD-75087 A-150680 UUCCCUCUCAACAAAACUUdTdT2824 A-150681 AAGUUUUGUUGAGAGGGAAdTdT 3104 UUCCCUCUCAACAAAACUU 3384AD-75088 A-150682 UUUAUAGGCAGUCUUCUGAdTdT 2825 A-150683UCAGAAGACUGCCUAUAAAdTdT 3105 UUUAUAGGCAGUCUUCUGC 3385 AD-75089 A-150684UUUUCUGUCAUAGUUAGAUdTdT 2826 A-150685 AUCUAACUAUGACAGAAAAdTdT 3106UUUUCUGUCAUAGUUAGAU 3386 AD-75090 A-150686 AUGUGAUAAUUCUAAGAGUdTdT 2827A-150687 ACUCUUAGAAUUAUCACAUdTdT 3107 AUGUGAUAAUUCUAAGAGU 3387 AD-75091A-150688 UUCCUUCACUUAAUUCUAUdTdT 2828 A-150689 AUAGAAUUAAGUGAAGGAAdTdT3108 UUCCUUCACUUAAUUCUAU 3388 AD-75092 A-150690 AUUAUCUUUCUUAACUUUUdTdT2829 A-150691 AAAAGUUAAGAAAGAUAAUdTdT 3109 AUUAUCUUUCUUAACUUUU 3389AD-75093 A-150692 UUCCAACACAUAAUCCUCUdTdT 2830 A-150693AGAGGAUUAUGUGUUGGAAdTdT 3110 UUCCAACACAUAAUCCUCU 3390 AD-75094 A-150694AAAUAAAUUGAAAAUAACUdTdT 2831 A-150695 AGUUAUUUUCAAUUUAUUUdTdT 3111AAAUAAAUUGAAAAUAACU 3391 AD-75095 A-150696 UCAUUAUACCAAUUCACUAdTdT 2832A-150697 UAGUGAAUUGGUAUAAUGAdTdT 3112 UCAUUAUACCAAUUCACUA 3392 AD-75096A-150698 AUUUUAUUUUUUAAUGAAUdTdT 2833 A-150699 AUUCAUUAAAAAAUAAAAUdTdT3113 AUUUUAUUUUUUAAUGAAU 3393 AD-75097 A-150700 UUAAAACUAGAAAACAAAUdTdT2834 A-150701 AUUUGUUUUCUAGUUUUAAdTdT 3114 UUAAAACUAGAAAACAAAU 3394AD-75098 A-150702 UUGAUUACUAUAUACUACAdTdT 2835 A-150703UGUAGUAUAUAGUAAUCAAdTdT 3115 UUGAUUACUAUAUACUACA 3395 AD-75099 A-150704AUGACUCAGAUUUCAUAGAdTdT 2836 A-150705 UCUAUGAAAUCUGAGUCAUdTdT 3116AUGACUCAGAUUUCAUAGA 3396 AD-75100 A-150706 AAAGGAGCAACCAAAAUGUdTdT 2837A-150707 ACAUUUUGGUUGCUCCUUUdTdT 3117 AAAGGAGCAACCAAAAUGU 3397 AD-75101A-150708 GUCACAACCCAAAACUUUAdTdT 2838 A-150709 UAAAGUUUUGGGUUGUGACdTdT3118 GUCACAACCCAAAACUUUA 3398 AD-75102 A-150710 AAACUUUACAAGCUUUGCUdTdT2839 A-150711 AGCAAAGCUUGUAAAGUUUdTdT 3119 AAACUUUACAAGCUUUGCU 3399AD-75103 A-150712 UUCAGAAUUAGAUUGCUUUdTdT 2840 A-150713AAAGCAAUCUAAUUCUGAAdTdT 3120 UUCAGAAUUAGAUUGCUUU 3400 AD-75104 A-150714UUAUAAUUCUUGAAUGAGAdTdT 2841 A-150715 UCUCAUUCAAGAAUUAUAAdTdT 3121UUAUAAUUCUUGAAUGAGG 3401 AD-75105 A-150716 UAAUUCUUGAAUGAGGCAAdTdT 2842A-150717 UUGCCUCAUUCAAGAAUUAdTdT 3122 UAAUUCUUGAAUGAGGCAA 3402 AD-75106A-150718 AUUUCAAGAUAUUUGUAAAdTdT 2843 A-150719 UUUACAAAUAUCUUGAAAUdTdT3123 AUUUCAAGAUAUUUGUAAA 3403 AD-75107 A-150720 AAGAACAGUAAACAUUGGUdTdT2844 A-150721 ACCAAUGUUUACUGUUCUUdTdT 3124 AAGAACAGUAAACAUUGGU 3404AD-75108 A-150722 UUUCAACUCAUAGGCUUAUdTdT 2845 A-150723AUAAGCCUAUGAGUUGAAAdTdT 3125 UUUCAACUCAUAGGCUUAU 3405 AD-75109 A-150724UUGACCAUACUGGAUACUUdTdT 2846 A-150725 AAGUAUCCAGUAUGGUCAAdTdT 3126UUGACCAUACUGGAUACUU 3406 AD-75110 A-150726 UUUAAGAUGAGGCAGUUCAdTdT 2847A-150727 UGAACUGCCUCAUCUUAAAdTdT 3127 UUUAAGAUGAGGCAGUUCC 3407 AD-75111A-150728 CAUCAGAAUCCACUCUUCUdTdT 2848 A-150729 AGAAGAGUGGAUUCUGAUGdTdT3128 CAUCAGAAUCCACUCUUCU 3408 AD-75112 A-150730 UAGGGAUAUGAAAAUCUCUdTdT2849 A-150731 AGAGAUUUUCAUAUCCCUAdTdT 3129 UAGGGAUAUGAAAAUCUCU 3409AD-75113 A-150732 UUCACCCUAAGGAUCCAAUdTdT 2850 A-150733AUUGGAUCCUUAGGGUGAAdTdT 3130 UUCACCCUAAGGAUCCAAU 3410 AD-75114 A-150734AUGGAAUACUGAAAAGAAAdTdT 2851 A-150735 UUUCUUUUCAGUAUUCCAUdTdT 3131AUGGAAUACUGAAAAGAAA 3411 AD-75115 A-150736 GAAUACUGAAAAGAAAUCAdTdT 2852A-150737 UGAUUUCUUUUCAGUAUUCdTdT 3132 GAAUACUGAAAAGAAAUCA 3412 AD-75116A-150738 ACUUCCUUGAAAAUUUUAUdTdT 2853 A-150739 AUAAAAUUUUCAAGGAAGUdTdT3133 ACUUCCUUGAAAAUUUUAU 3413 AD-75117 A-150740 UUAAAAAACAAACAAACAAdTdT2854 A-150741 UUGUUUGUUUGUUUUUUAAdTdT 3134 UUAAAAAACAAACAAACAA 3414AD-75118 A-150742 AAACAAAAAGCCUGUCCAAdTdT 2855 A-150743UUGGACAGGCUUUUUGUUUdTdT 3135 AAACAAAAAGCCUGUCCAC 3415 AD-75119 A-150744UUUGUGUAGAUGAAACCAUdTdT 2856 A-150745 AUGGUUUCAUCUACACAAAdTdT 3136UUUGUGUAGAUGAAACCAU 3416 AD-75120 A-150746 UUGGGAGAAGGCUUAGAAUdTdT 2857A-150747 AUUCUAAGCCUUCUCCCAAdTdT 3137 UUGGGAGAAGGCUUAGAAU 3417 AD-75121A-150748 UAAAAGAUGUAGCACAUUUdTdT 2858 A-150749 AAAUGUGCUACAUCUUUUAdTdT3138 UAAAAGAUGUAGCACAUUU 3418 AD-75122 A-150750 UUAUUGUUUGGCCAGCUAUdTdT2859 A-150751 AUAGCUGGCCAAACAAUAAdTdT 3139 UUAUUGUUUGGCCAGCUAU 3419AD-75123 A-150752 AUGCCAAUGUGGUGCUAUUdTdT 2860 A-150753AAUAGCACCACAUUGGCAUdTdT 3140 AUGCCAAUGUGGUGCUAUU 3420 AD-75124 A-150754AAUGUGGUGCUAUUGUUUAdTdT 2861 A-150755 UAAACAAUAGCACCACAUUdTdT 3141AAUGUGGUGCUAUUGUUUC 3421 AD-75125 A-150756 CUUUAAGAAAGUACUUGAAdTdT 2862A-150757 UUCAAGUACUUUCUUAAAGdTdT 3142 CUUUAAGAAAGUACUUGAC 3422 AD-75126A-150758 CUAAAAAAAAAAGAAAAAAdTdT 2863 A-150759 UUUUUUCUUUUUUUUUUAGdTdT3143 CUAAAAAAAAAAGAAAAAA 3423 AD-75127 A-150760 AAGAAAAAAAAGAAAGCAUdTdT2864 A-150761 AUGCUUUCUUUUUUUUCUUdTdT 3144 AAGAAAAAAAAGAAAGCAU 3424AD-75128 A-150762 AUAGACAUAUUUUUUUAAAdTdT 2865 A-150763UUUAAAAAAAUAUGUCUAUdTdT 3145 AUAGACAUAUUUUUUUAAA 3425 AD-75129 A-150764UUAAAGUAUAAAAACAACAdTdT 2866 A-150765 UGUUGUUUUUAUACUUUAAdTdT 3146UUAAAGUAUAAAAACAACA 3426 AD-75130 A-150766 CAAUUCUAUAGAUAGAUGAdTdT 2867A-150767 UCAUCUAUCUAUAGAAUUGdTdT 3147 CAAUUCUAUAGAUAGAUGG 3427 AD-75131A-150768 GGCUUAAUAAAAUAGCAUUdTdT 2868 A-150769 AAUGCUAUUUUAUUAAGCCdTdT3148 GGCUUAAUAAAAUAGCAUU 3428 AD-75132 A-150770 UAAUAAAAUAGCAUUAGGUdTdT2869 A-150771 ACCUAAUGCUAUUUUAUUAdTdT 3149 UAAUAAAAUAGCAUUAGGU 3429AD-75133 A-150772 UAUCUAGCCACCACCACCUdTdT 2870 A-150773AGGUGGUGGUGGCUAGAUAdTdT 3150 UAUCUAGCCACCACCACCU 3430 AD-75134 A-150774UUUAUCACUCACAAGUAGUdTdT 2871 A-150775 ACUACUUGUGAGUGAUAAAdTdT 3151UUUAUCACUCACAAGUAGU 3431 AD-75135 A-150776 GGCAGGAGUUGGAAAUUUUdTdT 2872A-150777 AAAAUUUCCAACUCCUGCCdTdT 3152 GGCAGGAGUUGGAAAUUUU 3432 AD-75136A-150778 UUUAAAGUUAGAAGGCUCAdTdT 2873 A-150779 UGAGCCUUCUAACUUUAAAdTdT3153 UUUAAAGUUAGAAGGCUCC 3433 AD-75137 A-150780 CCAUUGUUUUGUUGGCUCUdTdT2874 A-150781 AGAGCCAACAAAACAAUGGdTdT 3154 CCAUUGUUUUGUUGGCUCU 3434AD-75138 A-150782 UUAGCAAAAUUAGCAAUAUdTdT 2875 A-150783AUAUUGCUAAUUUUGCUAAdTdT 3155 UUAGCAAAAUUAGCAAUAU 3435 AD-75139 A-150784AUAUUAUCCAAUCUUCUGAdTdT 2876 A-150785 UCAGAAGAUUGGAUAAUAUdTdT 3156AUAUUAUCCAAUCUUCUGA 3436 AD-75140 A-150786 UUAUCCAAUCUUCUGAACUdTdT 2877A-150787 AGUUCAGAAGAUUGGAUAAdTdT 3157 UUAUCCAAUCUUCUGAACU 3437 AD-75141A-150788 AAGAGCAUGGAGAAUAAACdTdT 2878 A-150789 GUUUAUUCUCCAUGCUCUUdTdT3158 AAGAGCAUGGAGAAUAAAC 3438 AD-75142 A-150790 ACGCGGGAAAAAAGAUCUUdTdT2879 A-150791 AAGAUCUUUUUUCCCGCGUdTdT 3159 ACGCGGGAAAAAAGAUCUU 3439AD-75143 A-150792 GAUCUUAUAGGCAAAUAGAdTdT 2880 A-150793UCUAUUUGCCUAUAAGAUCdTdT 3160 GAUCUUAUAGGCAAAUAGA 3440 AD-75144 A-150794AAGAAUUUAAAAGAUAAGUdTdT 2881 A-150795 ACUUAUCUUUUAAAUUCUUdTdT 3161AAGAAUUUAAAAGAUAAGU 3441 AD-75145 A-150796 GUAAGUUCCUUAUUGAUUUdTdT 2882A-150797 AAAUCAAUAAGGAACUUACdTdT 3162 GUAAGUUCCUUAUUGAUUU 3442 AD-75146A-150798 UUUUGUGCACUCUGCUCUAdTdT 2883 A-150799 UAGAGCAGAGUGCACAAAAdTdT3163 UUUUGUGCACUCUGCUCUA 3443 AD-75147 A-150800 AAACAGAUAUUCAGCAAGUdTdT2884 A-150801 ACUUGCUGAAUAUCUGUUUdTdT 3164 AAACAGAUAUUCAGCAAGU 3444AD-75148 A-150802 UCAGCAAGUGGAGAAAAUAdTdT 2885 A-150803UAUUUUCUCCACUUGCUGAdTdT 3165 UCAGCAAGUGGAGAAAAUA 3445 AD-75149 A-150804AAGAACAAAGAGAAAAAAUdTdT 2886 A-150805 AUUUUUUCUCUUUGUUCUUdTdT 3166AAGAACAAAGAGAAAAAAU 3446 AD-75150 A-150806 AUACAUAGAUUUACCUGCAdTdT 2887A-150807 UGCAGGUAAAUCUAUGUAUdTdT 3167 AUACAUAGAUUUACCUGCA 3447 AD-75151A-150808 UUACCUGCAAAAAAUAGCUdTdT 2888 A-150809 AGCUAUUUUUUGCAGGUAAdTdT3168 UUACCUGCAAAAAAUAGCU 3448 AD-75152 A-150810 UUUAUAGAAGACAUUCUCAdTdT2889 A-150811 UGAGAAUGUCUUCUAUAAAdTdT 3169 UUUAUAGAAGACAUUCUCC 3449AD-75153 A-150812 AGACAUCUCAAAGAGCAGUdTdT 2890 A-150813ACUGCUCUUUGAGAUGUCUdTdT 3170 AGACAUCUCAAAGAGCAGU 3450 AD-75154 A-150814UAUGAGAUGGGGGUUAUCUdTdT 2891 A-150815 AGAUAACCCCCAUCUCAUAdTdT 3171UAUGAGAUGGGGGUUAUCU 3451 AD-75155 A-150816 CUACUGAUAAAGAAAGAAUdTdT 2892A-150817 AUUCUUUCUUUAUCAGUAGdTdT 3172 CUACUGAUAAAGAAAGAAU 3452 AD-75156A-150818 AAAGAAUUUAUGAGAAAUUdTdT 2893 A-150819 AAUUUCUCAUAAAUUCUUUdTdT3173 AAAGAAUUUAUGAGAAAUU 3453 AD-75157 A-150820 UAACAAUCUGUGAAGAUUUdTdT2894 A-150821 AAAUCUUCACAGAUUGUUAdTdT 3174 UAACAAUCUGUGAAGAUUU 3454AD-75158 A-150822 UUUUACUUUAUACAGUCUUdTdT 2895 A-150823AAGACUGUAUAAAGUAAAAdTdT 3175 UUUUACUUUAUACAGUCUU 3455 AD-75159 A-150824UUUAUGAAUUUCUUAAUGUdTdT 2896 A-150825 ACAUUAAGAAAUUCAUAAAdTdT 3176UUUAUGAAUUUCUUAAUGU 3456 AD-75160 A-150826 UUAAUGUUCAAAAUGACUUdTdT 2897A-150827 AAGUCAUUUUGAACAUUAAdTdT 3177 UUAAUGUUCAAAAUGACUU 3457 AD-75161A-150828 UUCUUCUUUUUUUAUAUCAdTdT 2898 A-150829 UGAUAUAAAAAAAGAAGAAdTdT3178 UUCUUCUUUUUUUAUAUCA 3458 AD-75162 A-150830 AGAAUGAGGAAUAAUAAGUdTdT2899 A-150831 ACUUAUUAUUCCUCAUUCUdTdT 3179 AGAAUGAGGAAUAAUAAGU 3459AD-75163 A-150832 UUAAACCCACAUAGACUCUdTdT 2900 A-150833AGAGUCUAUGUGGGUUUAAdTdT 3180 UUAAACCCACAUAGACUCU 3460 AD-75164 A-150834CUUUAAAACUAUAGGCUAAdTdT 2901 A-150835 UUAGCCUAUAGUUUUAAAGdTdT 3181CUUUAAAACUAUAGGCUAG 3461 AD-75165 A-150836 AGAUAGAAAUGUAUGUUUAdTdT 2902A-150837 UAAACAUACAUUUCUAUCUdTdT 3182 AGAUAGAAAUGUAUGUUUG 3462 AD-75166A-150838 UUUGACUUGUUGAAGCUAUdTdT 2903 A-150839 AUAGCUUCAACAAGUCAAAdTdT3183 UUUGACUUGUUGAAGCUAU 3463 AD-75167 A-150840 UUUUUAAUCUUAAAAGAUUdTdT2904 A-150841 AAUCUUUUAAGAUUAAAAAdTdT 3184 UUUUUAAUCUUAAAAGAUU 3464AD-75168 A-150842 UUGUGCUAAUUUAUUAGAAdTdT 2905 A-150843UUCUAAUAAAUUAGCACAAdTdT 3185 UUGUGCUAAUUUAUUAGAG 3465 AD-75169 A-150844UUAUUAGAGCAGAACCUGUdTdT 2906 A-150845 ACAGGUUCUGCUCUAAUAAdTdT 3186UUAUUAGAGCAGAACCUGU 3466 AD-75170 A-150846 GUUUGGCUCUCCUCAGAAAdTdT 2907A-150847 UUUCUGAGGAGAGCCAAACdTdT 3187 GUUUGGCUCUCCUCAGAAG 3467 AD-75171A-150848 CAAUAUUUUCAAAAGAUAAdTdT 2908 A-150849 UUAUCUUUUGAAAAUAUUGdTdT3188 CAAUAUUUUCAAAAGAUAA 3468 AD-75172 A-150850 UCAAAAGAUAAAUCUGAUUdTdT2909 A-150851 AAUCAGAUUUAUCUUUUGAdTdT 3189 UCAAAAGAUAAAUCUGAUU 3469AD-75173 A-150852 UUAUGCAAUGGCAUCAUUUdTdT 2910 A-150853AAAUGAUGCCAUUGCAUAAdTdT 3190 UUAUGCAAUGGCAUCAUUU 3470 AD-75174 A-150854UGCAAUGGCAUCAUUUAUUdTdT 2911 A-150855 AAUAAAUGAUGCCAUUGCAdTdT 3191UGCAAUGGCAUCAUUUAUU 3471 AD-75175 A-150856 UUUAAAACAGAAGAAUUGUdTdT 2912A-150857 ACAAUUCUUCUGUUUUAAAdTdT 3192 UUUAAAACAGAAGAAUUGU 3472 AD-75176A-150858 AACAACAAAAGGAAAAUGUdTdT 2913 A-150859 ACAUUUUCCUUUUGUUGUUdTdT3193 AACAACAAAAGGAAAAUGU 3473 AD-75177 A-150860 UUAAUCCUGUAGUACAUAUdTdT2914 A-150861 AUAUGUACUACAGGAUUAAdTdT 3194 UUAAUCCUGUAGUACAUAU 3474AD-75178 A-150862 UUUAAUAUUUUAUAAGACAdTdT 2915 A-150863UGUCUUAUAAAAUAUUAAAdTdT 3195 UUUAAUAUUUUAUAAGACC 3475 AD-75179 A-150864CCUUCCUGUUAGGUAUUAAdTdT 2916 A-150865 UUAAUACCUAACAGGAAGGdTdT 3196CCUUCCUGUUAGGUAUUAG 3476 AD-75180 A-150866 UUAGGUAUUAGAAAGUGAUdTdT 2917A-150867 AUCACUUUCUAAUACCUAAdTdT 3197 UUAGGUAUUAGAAAGUGAU 3477 AD-75181A-150868 AUACAUAGAUAUCUUUUUUdTdT 2918 A-150869 AAAAAAGAUAUCUAUGUAUdTdT3198 AUACAUAGAUAUCUUUUUU 3478 AD-75182 A-150870 UUUUUGUGUAAUUUCUAUUdTdT2919 A-150871 AAUAGAAAUUACACAAAAAdTdT 3199 UUUUUGUGUAAUUUCUAUU 3479AD-75183 A-150872 UUAAAAAAGAGAGAAGACUdTdT 2920 A-150873AGUCUUCUCUCUUUUUUAAdTdT 3200 UUAAAAAAGAGAGAAGACU 3480 AD-75184 A-150874CUGUCAGAAGCUUUAAGUAdTdT 2921 A-150875 UACUUAAAGCUUCUGACAGdTdT 3201CUGUCAGAAGCUUUAAGUG 3481 AD-75185 A-150876 UAUGGUACAGGAUAAAGAUdTdT 2922A-150877 AUCUUUAUCCUGUACCAUAdTdT 3202 UAUGGUACAGGAUAAAGAU 3482 AD-75186A-150878 UUAAAUAACCAAUUCCUAUdTdT 2923 A-150879 AUAGGAAUUGGUUAUUUAAdTdT3203 UUAAAUAACCAAUUCCUAU 3483 AD-75187 A-150880 UUGUUUUUUAAAGAAACCUdTdT2924 A-150881 AGGUUUCUUUAAAAAACAAdTdT 3204 UUGUUUUUUAAAGAAACCU 3484AD-75188 A-150882 CUCUCACAGAUAAGACAGAdTdT 2925 A-150883UCUGUCUUAUCUGUGAGAGdTdT 3205 CUCUCACAGAUAAGACAGA 3485 AD-75189 A-150884CAGAAUUUUAUAGAGGGCUdTdT 2926 A-150885 AGCCCUCUAUAAAAUUCUGdTdT 3206CAGAAUUUUAUAGAGGGCU 3486 AD-75190 A-150886 UCUAGAAUUAAAGGAACCUdTdT 2927A-150887 AGGUUCCUUUAAUUCUAGAdTdT 3207 UCUAGAAUUAAAGGAACCU 3487 AD-75191A-150888 CUCACUGAAAACAUAUAUUdTdT 2928 A-150889 AAUAUAUGUUUUCAGUGAGdTdT3208 CUCACUGAAAACAUAUAUU 3488 AD-75192 A-150890 AAACAUAUAUUUCACGUGUdTdT2929 A-150891 ACACGUGAAAUAUAUGUUUdTdT 3209 AAACAUAUAUUUCACGUGU 3489AD-75193 A-150892 GUUCCCUCUUUUUUUUUUUdTdT 2930 A-150893AAAAAAAAAAAGAGGGAACdTdT 3210 GUUCCCUCUUUUUUUUUUU 3490 AD-75194 A-150894UUAAGCGAUUCUCCUGCCUdTdT 2931 A-150895 AGGCAGGAGAAUCGCUUAAdTdT 3211UUAAGCGAUUCUCCUGCCU 3491 AD-75195 A-150896 CGGCUAAUUUUUUGGAUUUdTdT 2932A-150897 AAAUCCAAAAAAUUAGCCGdTdT 3212 CGGCUAAUUUUUUGGAUUU 3492 AD-75196A-150898 UUUAAUAGAGACGGGGUUUdTdT 2933 A-150899 AAACCCCGUCUCUAUUAAAdTdT3213 UUUAAUAGAGACGGGGUUU 3493 AD-75197 A-150900 UUUACCAUGUUGGCCAGGUdTdT2934 A-150901 ACCUGGCCAACAUGGUAAAdTdT 3214 UUUACCAUGUUGGCCAGGU 3494AD-75198 A-150902 UUGCUGGGAUUACAGGCAUdTdT 2935 A-150903AUGCCUGUAAUCCCAGCAAdTdT 3215 UUGCUGGGAUUACAGGCAU 3495 AD-75199 A-150904UUAAACAUGAUCCUUCUCUdTdT 2936 A-150905 AGAGAAGGAUCAUGUUUAAdTdT 3216UUAAACAUGAUCCUUCUCU 3496 AD-75200 A-150906 GGGGUCUUUCAAGGGGAAAdTdT 2937A-150907 UUUCCCCUUGAAAGACCCCdTdT 3217 GGGGUCUUUCAAGGGGAAA 3497 AD-75201A-150908 AAAAAUCCAAGCUUUUUUAdTdT 2938 A-150909 UAAAAAAGCUUGGAUUUUUdTdT3218 AAAAAUCCAAGCUUUUUUA 3498 AD-75202 A-150910 AAAGUAAAAAAAAAAAAAGdTdT2939 A-150911 CUUUUUUUUUUUUUACUUUdTdT 3219 AAAGUAAAAAAAAAAAAAG 3499AD-75203 A-150912 AGAGAGGACACAAAACCAAdTdT 2940 A-150913UUGGUUUUGUGUCCUCUCUdTdT 3220 AGAGAGGACACAAAACCAA 3500 AD-75204 A-150914UUAAGAUGGAGACAGAGUUdTdT 2941 A-150915 AACUCUGUCUCCAUCUUAAdTdT 3221UUAAGAUGGAGACAGAGUU 3501 AD-75205 A-150916 UUUCUCCUAAUAACCGGAAdTdT 2942A-150917 UUCCGGUUAUUAGGAGAAAdTdT 3222 UUUCUCCUAAUAACCGGAG 3502 AD-75206A-150918 GCUGAAUUACCUUUCACUUdTdT 2943 A-150919 AAGUGAAAGGUAAUUCAGCdTdT3223 GCUGAAUUACCUUUCACUU 3503 AD-75207 A-150920 UUCAAAAACAUGACCUUCAdTdT2944 A-150921 UGAAGGUCAUGUUUUUGAAdTdT 3224 UUCAAAAACAUGACCUUCC 3504AD-75208 A-150922 CAAUCCUUAGAAUCUGCCUdTdT 2945 A-150923AGGCAGAUUCUAAGGAUUGdTdT 3225 CAAUCCUUAGAAUCUGCCU 3505 AD-75209 A-150924UUUUAUAUUACUGAGGCCUdTdT 2946 A-150925 AGGCCUCAGUAAUAUAAAAdTdT 3226UUUUAUAUUACUGAGGCCU 3506 AD-75210 A-150926 AAAAGUAAACAUUACUCAUdTdT 2947A-150927 AUGAGUAAUGUUUACUUUUdTdT 3227 AAAAGUAAACAUUACUCAU 3507 AD-75211A-150928 UUUAUUUUGCCCAAAAUGAdTdT 2948 A-150929 UCAUUUUGGGCAAAAUAAAdTdT3228 UUUAUUUUGCCCAAAAUGC 3508 AD-75212 A-150930 CACUGAUGUAAAGUAGGAAdTdT2949 A-150931 UUCCUACUUUACAUCAGUGdTdT 3229 CACUGAUGUAAAGUAGGAA 3509AD-75213 A-150932 AAAAUAAAAACAGAGCUCUdTdT 2950 A-150933AGAGCUCUGUUUUUAUUUUdTdT 3230 AAAAUAAAAACAGAGCUCU 3510 AD-75214 A-150934CUAAAAUCCCUUUCAAGCAdTdT 2951 A-150935 UGCUUGAAAGGGAUUUUAGdTdT 3231CUAAAAUCCCUUUCAAGCC 3511 AD-75215 A-150936 UUGACCCCACUCACCAACUdTdT 2952A-150937 AGUUGGUGAGUGGGGUCAAdTdT 3232 UUGACCCCACUCACCAACU 3512 AD-75216A-150938 UUAUCUUGUACCCGCUGCUdTdT 2953 A-150939 AGCAGCGGGUACAAGAUAAdTdT3233 UUAUCUUGUACCCGCUGCU 3513 AD-75217 A-150940 CUGAAACCUCAAGCUGUCUdTdT2954 A-150941 AGACAGCUUGAGGUUUCAGdTdT 3234 CUGAAACCUCAAGCUGUCU 3514AD-75218 A-150942 GUAUCAUGAAAAUGUCUAUdTdT 2955 A-150943AUAGACAUUUUCAUGAUACdTdT 3235 GUAUCAUGAAAAUGUCUAU 3515 AD-75219 A-150944UUCAAAAUAUCAAAACCUUdTdT 2956 A-150945 AAGGUUUUGAUAUUUUGAAdTdT 3236UUCAAAAUAUCAAAACCUU 3516 AD-75220 A-150946 UUUCAAAUAUCACGCAGCUdTdT 2957A-150947 AGCUGCGUGAUAUUUGAAAdTdT 3237 UUUCAAAUAUCACGCAGCU 3517 AD-75221A-150948 CUUAUAUUCAGUUUACAUAdTdT 2958 A-150949 UAUGUAAACUGAAUAUAAGdTdT3238 CUUAUAUUCAGUUUACAUA 3518 AD-75222 A-150950 UUACAUAAAGGCCCCAAAUdTdT2959 A-150951 AUUUGGGGCCUUUAUGUAAdTdT 3239 UUACAUAAAGGCCCCAAAU 3519AD-75223 A-150952 AUACCAUGUCAGAUCUUUUdTdT 2960 A-150953AAAAGAUCUGACAUGGUAUdTdT 3240 AUACCAUGUCAGAUCUUUU 3520 AD-75224 A-150954AAAAGAGUUAAUGAACUAUdTdT 2961 A-150955 AUAGUUCAUUAACUCUUUUdTdT 3241AAAAGAGUUAAUGAACUAU 3521 AD-75225 A-150956 AUGAGAAUUGGGAUUACAUdTdT 2962A-150957 AUGUAAUCCCAAUUCUCAUdTdT 3242 AUGAGAAUUGGGAUUACAU 3522 AD-75226A-150958 AUCAUGUAUUUUGCCUCAUdTdT 2963 A-150959 AUGAGGCAAAAUACAUGAUdTdT3243 AUCAUGUAUUUUGCCUCAU 3523 AD-75227 A-150960 UUAUCACACUUAUAGGCCAdTdT2964 A-150961 UGGCCUAUAAGUGUGAUAAdTdT 3244 UUAUCACACUUAUAGGCCA 3524AD-75228 A-150962 CAAGUGUGAUAAAUAAACUdTdT 2965 A-150963AGUUUAUUUAUCACACUUGdTdT 3245 CAAGUGUGAUAAAUAAACU 3525 AD-75229 A-150964UUACAGACACUGAAUUAAUdTdT 2966 A-150965 AUUAAUUCAGUGUCUGUAAdTdT 3246UUACAGACACUGAAUUAAU 3526 AD-75230 A-150966 UUUGAAACCAGAAAAUAAUdTdT 2967A-150967 AUUAUUUUCUGGUUUCAAAdTdT 3247 UUUGAAACCAGAAAAUAAU 3527 AD-75231A-150968 AUGACUGGCCAUUCGUUAAdTdT 2968 A-150969 UUAACGAAUGGCCAGUCAUdTdT3248 AUGACUGGCCAUUCGUUAC 3528 AD-75232 A-150970 UUAGUUGAAAAGCAUAUUUdTdT2969 A-150971 AAAUAUGCUUUUCAACUAAdTdT 3249 UUAGUUGAAAAGCAUAUUU 3529AD-75233 A-150972 UUUUUAUUAAAUUAAUUCUdTdT 2970 A-150973AGAAUUAAUUUAAUAAAAAdTdT 3250 UUUUUAUUAAAUUAAUUCU 3530 AD-75234 A-150974CUGAUUGUAUUUGAAAUUAdTdT 2971 A-150975 UAAUUUCAAAUACAAUCAGdTdT 3251CUGAUUGUAUUUGAAAUUA 3531 AD-75235 A-150976 UUUGAAAUUAUUAUUCAAUdTdT 2972A-150977 AUUGAAUAAUAAUUUCAAAdTdT 3252 UUUGAAAUUAUUAUUCAAU 3532 AD-75236A-150978 UUAUGGCAGAGGAAUAUCAdTdT 2973 A-150979 UGAUAUUCCUCUGCCAUAAdTdT3253 UUAUGGCAGAGGAAUAUCA 3533 AD-75237 A-150980 UCUAAAAAUGUAACUAAUUdTdT2974 A-150981 AAUUAGUUACAUUUUUAGAdTdT 3254 UCUAAAAAUGUAACUAAUU 3534AD-75238 A-150982 UUUACUGUUUAAUAAGCAUdTdT 2975 A-150983AUGCUUAUUAAACAGUAAAdTdT 3255 UUUACUGUUUAAUAAGCAU 3535 AD-75239 A-150984UGUCAUAAUAAAAUGGUAUdTdT 2976 A-150985 AUACCAUUUUAUUAUGACAdTdT 3256UGUCAUAAUAAAAUGGUAU 3536 AD-75240 A-150986 AUAUCUUUCUUUAGUAAUUdTdT 2977A-150987 AAUUACUAAAGAAAGAUAUdTdT 3257 AUAUCUUUCUUUAGUAAUU 3537 AD-75241A-150988 UUAGUAAUUACAUUAAAAUdTdT 2978 A-150989 AUUUUAAUGUAAUUACUAAdTdT3258 UUAGUAAUUACAUUAAAAU 3538 AD-75242 A-150990 AUUAGUCAUGUUUGAUUAAdTdT2979 A-150991 UUAAUCAAACAUGACUAAUdTdT 3259 AUUAGUCAUGUUUGAUUAA 3539

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent forinhibiting expression of an insulin-like growth factor binding protein,acid labile subunit (IGFALS) gene, wherein the dsRNA agent comprises asense strand and an antisense strand forming a double stranded region,wherein said antisense strand comprises at least 17 contiguousnucleotides of the nucleotide sequence of 5′-AGUGAGUUGUUCCUGAGGCUGAG-3′(SEQ ID NO: 135), and wherein the dsRNA agent comprises at least onemodified nucleotide.
 2. The dsRNA agent of claim 1, wherein thenucleotide sequence of the antisense strand is5′-AGUGAGUUGUUCCUGAGGCUGAG-3′ (SEQ ID NO: 135).
 3. The dsRNA agent ofclaim 2, wherein the nucleotide sequence of the antisense strand is5′-AGUGAGUUGUUCCUGAGGCUGAG-3′ (SEQ ID NO: 135) and the nucleotidesequence of the sense strand is 5′-CAGCCUCAGGAACAACUCACU-3′ (SEQ ID NO:78).
 4. The dsRNA agent of claim 1, wherein substantially all of thenucleotides of said sense strand or substantially all of the nucleotidesof said antisense strand comprise a nucleotide modification.
 5. ThedsRNA agent of claim 1, wherein all of the nucleotides of said sensestrand and all of the nucleotides of said antisense strand comprise anucleotide modification.
 6. The dsRNA agent of claim 4, wherein at leastone of the nucleotides comprising a nucleotide modification comprises anucleotide modification selected from the group consisting of adeoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide, a conformationally restricted nucleotide, a constrainedethyl nucleotide, an a basic nucleotide, a 2′-amino-modified nucleotide,a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic.
 7. The dsRNA agent of claim 1, whereinthe antisense strand is 19-21 nucleotides in length.
 8. The dsRNA agentof claim 1, wherein each strand is no more than 30 nucleotides inlength.
 9. The dsRNA agent of claim 1, wherein at least one strandcomprises a 3′ overhang of at least 1 nucleotide; or at least 2nucleotides.
 10. The dsRNA agent of claim 1, further comprising aligand.
 11. The dsRNA agent of claim 10, wherein the ligand isconjugated to the 3′ end of the sense strand of the dsRNA agent.
 12. ThedsRNA agent of claim 10, wherein the ligand is an N-acetylgalactosamine(GalNAc) derivative.
 13. The dsRNA agent of claim 12, wherein the ligandis


14. The dsRNA agent of claim 12, wherein the dsRNA agent is conjugatedto the ligand as shown in the following schematic

and, wherein X is O or S.
 15. The dsRNA agent of claim 14, wherein the Xis O.
 16. The dsRNA agent of claim 1, wherein the double stranded regionis 17-23 nucleotide pairs in length.
 17. The dsRNA agent of claim 1,wherein each strand is independently 17-30 nucleotides in length. 18.The dsRNA agent of claim 1, wherein the at least one modified nucleotidecomprises a nucleotide modification selected from the group consistingof LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl,2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.
 19. ThedsRNA agent of claim 1, wherein said agent further comprises at leastone phosphorothioate or methylphosphonate internucleotide linkage.
 20. Apharmaceutical composition for inhibiting expression of an IGFALS genecomprising the dsRNA agent of claim
 1. 21. The dsRNA agent of claim 1,wherein the sense strand comprises the nucleotide sequence of5′-csasgccuCfaGfGfAfacaacucacuL96-3′ (SEQ ID NO: 184) and the antisensestrand comprises the nucleotide sequence of5′-asGfsugaGfuUfGfuuccUfgAfggcugsasg-3′ (SEQ ID NO: 241), wherein Af is2′-fluoroadenosine-3′-phosphate, Cf is 2′-fluorocytidine-3′-phosphate,Gf is 2′-fluoroguanosine-3′-phosphate, and Uf is2′-fluorouridine-3′-phosphate; wherein a is2′-O-methyladenosine-3′-phosphate, c is2′-O-methylcytidine-3′-phosphate, g is2′-O-methylguanosine-3′-phosphate, and u is2′-O-methyluridine-3′-phosphate; wherein s is a phosphorothioatelinkage; and wherein L96 isN-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
 22. The dsRNAagent of claim 1, wherein the sense strand consists of the nucleotidesequence of 5′-csasgccuCfaGfGfAfacaacucacuL96-3′ (SEQ ID NO: 184) andthe antisense strand consists of the nucleotide sequence of5′-asGfsugaGfuUfGfuuccUfgAfggcugsasg-3′ (SEQ ID NO: 241), wherein Af is2′-fluoroadenosine-3′-phosphate, Cf is 2′-fluorocytidine-3′-phosphate,Gf is 2′-fluoroguanosine-3′-phosphate, and Uf is2′-fluorouridine-3′-phosphate; wherein a is2′-O-methyladenosine-3′-phosphate, c is2′-O-methylcytidine-3′-phosphate, g is2′-O-methylguanosine-3′-phosphate, and u is2′-O-methyluridine-3′-phosphate; wherein s is a phosphorothioatelinkage; and wherein L96 isN-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.